Methods of treating patients having TLR3-positive cancer

ABSTRACT

The present invention relates to antibodies, antibody fragments, and derivatives thereof that specifically bind to TLR3 cell receptors present on the surface of cells. The invention also relates to hybridomas producing such antibodies; methods of making such antibodies; fragments, variants, and derivatives of the antibodies; pharmaceutical compositions comprising the same; methods of using the antibodies to detect TLR3 levels on the surface of cells, and the use of such antibodies and compositions for diagnostic or therapeutic purposes in subjects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/119,341, filed May11, 2011, now U.S. Pat. No. 8,273,541, which is the U.S. national stageapplication of International Patent Application No. PCT/EP2009/061902,filed Sep. 15, 2009, which claims the benefit of U.S. Provisional PatentApplication No. 61/097,676, filed Sep. 17, 2008, the disclosures ofwhich are hereby incorporated by reference in their entireties,including all figures, tables and amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Mar. 11, 2011 and is 19 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to antibodies, antibody fragments, andderivatives thereof that specifically bind to TLR3 cell receptorspresent on the surface of cells. The invention also relates tohybridomas producing such antibodies; methods of making such antibodies;fragments, variants, and derivatives of the antibodies; pharmaceuticalcompositions comprising the same; methods of using the antibodies todetect TLR3 levels on the surface of cells, and the use of suchantibodies and compositions for diagnostic or therapeutic purposes insubjects.

BACKGROUND

Cancer is a major cause of death in the world. Because traditionalcancer therapy targets all rapidly dividing cells, these therapies canhave devastating side effects because they affect non-cancerous cellssuch as cells of the gastrointestinal tract, immune system, and hairfollicles. Therefore, new methods of treatment are needed that are ableto more specifically target cancer cells and, as such, avoid the sideeffects typical of cancer therapy.

Drosophila toll proteins control dorsal-ventral patterning and arethought to represent an ancient host defense mechanism. In humans, TLRsare believed to be an important component of innate immunity. Human andDrosophila Toll protein sequences show homology over the entire lengthof the protein chains. The family of human Toll-like receptors iscomprised of ten highly conserved receptor proteins, TLR1-TLR10. LikeDrosophila toll, human TLRs are type I transmembrane proteins with anextracellular domain consisting of a leucine-rich repeat (LRR) domainthat recognizes pathogen-associated molecular patterns (PAMPs), and acytoplasmic domain that is homologous to the cytoplasmic domain of thehuman interleukin-1 (IL-1) receptor. Similar to the signaling pathwaysfor both Drosophila toll and the IL-1 receptor, human Toll-likereceptors signal through the NF-κB pathway.

Although the different mammalian TLRs share many characteristics andsignal transduction mechanisms, their biological functions are verydifferent. This is due in part to the fact that four different adaptormolecules (MyD88, TIRAP, TRIF and TRAF) are associated in variouscombinations with the TLRs and mediate different signaling pathways. Inaddition, different ligands for one TLR may preferentially activatedifferent signal transduction pathways. Furthermore, the TLRs aredifferentially expressed in various hematopoietic and non-hematopoieticcells. Accordingly, the response to a TLR ligand depends not only on thesignal pathway activated by the TLR, but also on the nature of the cellsin which the individual TLR is expressed.

Although ligands for some TLRs remain to be identified, a number of TLRspecific ligands have been reported. For example, TLR3 ligands(agonists) include double stranded RNA such as Poly IC and Poly AU.Polyinosinic-polycytidylic acid (Poly IC) is a high molecular weightsynthetic double stranded RNA that is heterogeneous in size.Polyadenylic-polyuridylic acid (Poly AU) is a double stranded complex ofsynthetic polyribonucleotides. Both Poly IC and Poly AU have been usedin several clinical trials as adjuvant therapy in different types ofcancer, such as cancer of the breast, bladder, kidney and stomach.

Stimulation of TLR3 by double stranded RNA or other agonists in, e.g.,dendritic cells or B lymphocytes leads to the production of cytokinessuch as IFN, the activation of the innate immune system (NK cells), theenhancement of CD8+ T cells, and antigen cross-priming by dendriticcells. Accordingly, TLR3 plays an important role in the defense againstviral infection, and agonists have been used as adjuvants for cancertherapy in the past (see, e.g., Lacour et al. (1980) Lancet 2: 161-164;Khan et al., (1995) Eur. J. Surg. Oncol. 21:224-227).

While most studies of human TLRs, e.g., TLR3, have focused on their rolein immune cells, it is now clear that they are also widely expressed innon-immune cells, including transformed cells such as breast cancer,cervical cancer, hepatomas, and melanomas, among others. Indeed, thereis evidence that the efficacy of TLR3 agonists such as poly-AU orpoly-IC in cancer therapy is not necessarily based on immune cellactivation but on the induction of apoptosis in TLR3-expressing tumorcells (see, e.g., Salaun et al. Clin Cancer Res (2007); 4565 13(15) Aug.1, 2007; Salaun et al., 2006, The Journal of Immunology, 176: 4894-4901;the entire disclosures of which are herein incorporated by reference).Because such TLR3 agonist therapy depends on the expression of TLR3 inthe tumor cells, therefore, it is of obvious utility to be able toreliably and easily detect TLR3 levels in tumor cells.

A simple and practical way of detecting the expression of specificproteins in vivo is by immunostaining of paraffin-embedded tissuesections. Using this method, thin sections of tissue (e.g., cancertissue obtained by biopsy) are obtained, fixed in e.g., formalin,embedded in paraffin, and then cut into very thin sections and mountedon slides. Following deparaffination, the slides are amenable to, e.g.,immunohistochemical methods to detect the expression of specificproteins. This method is particularly useful because it gives rise tostable preparations in which the specific cellular and intracellularlocalization of specific proteins can be assessed.

Unfortunately, however, it is not always possible to find antibodies,particularly monoclonal antibodies, that work effectively andspecifically in paraffin-embedded sections. Indeed, it is often muchmore difficult to obtain useful antibodies for immunohistochemistry onparaffin-embedded slides than it is for antibodies for use in otherdetection methods such as immunoblotting. The present inventionaddresses these and other needs.

SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods involvingantibodies, antibody fragments, and derivatives that allow specificbinding to human TLR3-expressing cells, particularly inparaffin-embedded sections. Such compositions and methods are useful fora multitude of applications, particularly for detecting TLR3 expressionand expression levels in cells or tissues, e.g., prior to TLR3-agonisttherapy. In addition, antibodies of the invention can be used forpurifying TLR3-expressing cells, for stimulating or inhibiting TLRreceptors and for specifically labeling TLR3-expressing cells in vitroor in vivo, e.g. for diagnostic or therapeutic purposes.

Accordingly, in one aspect, the present invention provides a monoclonalantibody that specifically binds a human TLR3 polypeptide, wherein saidantibody specifically binds to said TLR3 polypeptide inparaffin-embedded tissue sections. In one embodiment of the invention,the antibody competes for binding to the same TLR3 epitope as monoclonalantibody 40F9. In another embodiment, the antibody is a mouse antibody.In another embodiment, the isotype of the antibody is IgG, optionally anIgG1. In one embodiment, the antibody is chimeric, e.g. contains anon-murine, optionally a human, constant region. In another embodiment,the antibody does not substantially bind to human TLR4, e.g., inparaffin-embedded tissue sections. In another embodiment, the antibodycomprises a light chain comprising one, two or all three CDRs of the40F9 light chain variable region sequence of SEQ ID NO:3. In anotherembodiment, the antibody comprises a heavy chain comprising one, two orall three CDRs of the 40F9 heavy chain variable region sequence of SEQID NO:4. In another embodiment, the antibody comprises a light chainhaving a CDR comprising an amino acid sequence of any one of SEQ IDNOS:5 to 7. In another embodiment, the antibody comprises a heavy chainhaving a CDR comprising an amino acid sequence of any one of SEQ IDNOS:8 to 10. In another embodiment, the antibody is 40F9 or a fragmentor derivative thereof.

In another embodiment, the antibody is an antibody fragment selectedfrom Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, diabodies, single-chain antibodyfragment, or a multispecific antibody comprising multiple differentantibody fragments. In another embodiment, the antibody is conjugated orcovalently bound to a detectable moiety. In another embodiment, theantibody specifically binds to a healthy human tissue selected from thegroup consisting of skin, cerebellum, breast, lung, esophagus, stomach,ileum, jejunum, duodenum, colon, liver, pancreas, testis, spleen,thymus, and tonsil.

In another aspect, the present invention provides kits comprising any ofthe anti-TLR3 antibodies of the invention, preferably together withinstructions for their use, e.g., according to the therapeutic ordiagnostic methods provided herein. In one embodiment, the kit furthercomprises a labeled secondary antibody that specifically recognizes theprimary anti-TLR3 antibodies. In one such embodiment, the secondaryantibody is conjugated to HRP or AP. In another embodiment, the HRP orAP is conjugated to a polymer.

In another aspect, the present invention provides a cell, e.g. ahybridoma, producing an anti-TLR3 antibody of the invention. In oneembodiment, the cell is clone 40F9. In a related aspect, the presentinvention provides a hybridoma comprising: a) a B cell from a non-humanmammalian host that has been immunized with an antigen that comprisesthe TLR3 epitope specifically recognized by the 40F9 antibody, fused tob) an immortalized cell, wherein the hybridoma produces a monoclonalantibody that specifically binds to the epitope.

In one embodiment of either of these aspects, the monoclonal antibodybinds to the same epitope as antibody 40F9. In a particularly preferredembodiment, the hybridoma produces antibody 40F9.

In another aspect, the present invention provides a method of producingan antibody that specifically binds to TLR3 in paraffin-embeddedtissues, said method comprising the steps of: a) immunizing a non-humanmammal with an immunogen comprising a human TLR3 polypeptide; and b)preparing antibodies from said immunized animal that compete for bindingto said TLR3 polypeptide with antibody 40F9.

In one embodiment, the antibodies prepared in step (b) are monoclonalantibodies. In another embodiment, the method further comprise a step inwhich the ability of said antibodies to specifically bind to human TLR3polypeptides in paraffin-embedded tissue sections is assessed. In oneembodiment, the ability of the antibodies to bind to other TLR familymembers is assessed, optionally in paraffin-embedded tissue sections. Inone embodiment, the ability of the antibodies to bind to TLR4 isassessed. In another embodiment, the method further comprises the stepof making fragments or derivatives of the selected monoclonalantibodies. In one embodiment, the fragments or derivatives are selectedfrom the group consisting of Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, diabodies,single-chain antibody fragment, multispecific antibodies comprisingmultiple different antibody fragments, humanized antibodies, andchimeric antibodies. In another embodiment, the non-human mammal is amouse.

In another aspect, the present invention provides a method of detectingthe level of TLR3 in a paraffin-embedded tissue sample, the methodcomprising the steps of a) contacting the tissue sample with ananti-TLR3 antibody of the invention; and b) detecting the presence ofthe bound antibody in the tissue sample.

In one embodiment, the antibody is a monoclonal antibody that competesfor binding with antibody 40F9. In another embodiment, the antibody isantibody 40F9. In another embodiment, the tissue sample comprises ahuman tissue selected from the group consisting of skin, cerebellum,breast, lung, esophagus, stomach, ileum, jejunum, duodenum, colon,liver, pancreas, testis, spleen, thymus, and tonsil. In anotherembodiment, the tissue is a tumor tissue selected from the groupconsisting of breast, lung, esophagus, stomach, larynx, kidney, andcervix. In another embodiment, the tissue is breast tissue, and thepatient has breast cancer. In another embodiment, the tissue comprisesmelanoma cells. In another embodiment, the biological sample is takenfrom a patient, and the method is performed for diagnostic purposes. Inanother embodiment, the biological sample is taken from a healthyindividual.

In another embodiment, the antibody is conjugated or covalently bound toa detectable moiety. In another embodiment, the TLR3 levels are detectedusing a secondary antibody that specifically binds to said antibody. Inanother embodiment, the secondary antibody is covalently linked to HRPor AP. In another antibody, the HRP or AP is bound to a polymer.

In another aspect, the present invention provides a method of treating apatient with cancer, the method comprising a) providing aparaffin-embedded cancer tissue sample from the patient; b) detectingTLR3 levels in the tissue sample; and c) if TLR3 expression is detectedin the sample, administering a TLR3 ligand to the patient.

In one embodiment, the TLR3 levels are detected using a monoclonalantibody that competes for binding with antibody 40F9. In anotherembodiment, the antibody is antibody 40F9. In another embodiment, instep c) a TLR3 ligand is administered to the patient if the level ofTLR3 detected in step b) is elevated. In another embodiment, thecancerous tissue is selected from the group consisting of breast, lung,esophagus, stomach, larynx, kidney, and cervix. In another embodiment,the tissue is breast tissue, and the patient has breast cancer. Inanother embodiment, the tissue is a melanoma.

In another embodiment, the antibody is conjugated or covalently bound toa detectable moiety. In another embodiment, the TLR3 levels are detectedusing a secondary antibody that specifically binds to said antibody. Inanother embodiment, the secondary antibody is covalently linked to HRPor AP. In another embodiment, the TLR3 ligand is selected from the groupconsisting of double stranded RNA, poly I:C, and poly A:U.

In another embodiment, the method further comprises the step ofadministering to the patient an appropriate additional therapeutic agentselected from the group consisting of an immunomodulatory agent, ahormonal agent, a chemotherapeutic agent, an anti-angiogenic agent, anapoptotic agent, a targeting agent, and an adjunct compound.

These and additional advantageous aspects and features of the inventionmay be further described elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows results for commercial antibodies TLR3.7 from eBioscience,Inc. (graph 1) and goat pAb anti TLR3 from R&D Systems Inc. (graph 2),as tested in Biacore for binding to immobilized human TLR3 and TLR4protein. Binding is represented ordinates (in resonance units (RU)),time is represented in axis (in seconds). While both antibodies showedbinding to TLR3 over TLR4 protein in Biacore (binding on TLR3 chiprepresented in full line, binding on TLR4 chip represented in dottedline), these antibodies either showed no binding or were no longerspecific for TLR3 when tested in tissue sections as detailed in theexample section.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The present invention provides novel methods for producing and usingantibodies and other compounds suitable for the diagnosis and treatmentof disorders such as viral infection or cancer, particularly breastcancer, melanoma, cervical cancer, and hepatomas. Antibodies, antibodyderivatives, antibody fragments, and hybridomas are encompassed, as aremethods of producing the same and methods of treating and diagnosingpatients using the antibodies and compounds.

The present invention is based, in part, on the discovery thatmonoclonal antibodies can be generated, such antibodies bindingspecifically and efficiently to TLR3-expressing cells in fixed,paraffin-embedded tissue samples. Generally, monoclonal antibodiesdirected against human TLR3 are either incapable of binding to TLR3 inparaffin-embedded tissue samples and/or are not specific for TLR3 andthus also bind to other polypeptides, including other TLR polypeptidessuch as TLR4. Overcoming this limitation, the inventors have identifiedepitopes present on human TLR3, including the epitope recognized byantibody 40F9, that are particularly accessible to binding inparaffin-embedded sections.

Accordingly, the present invention provides monoclonal antibodies, andderivatives and fragments thereof, that specifically bind to human TLR3in paraffin-embedded tissue sections. In a preferred embodiment, theantibodies bind to the same epitope as, i.e. they compete for bindingto, the epitope recognized by antibody 40F9. In addition to antibodies,antibody fragments, antibody derivatives, hybridomas and other cells,and methods of making each of these, the present invention also providesmethods of detecting TLR3 levels in tissue and cell samples, as well asmethods of treating and diagnosing conditions such as viral infectionsand cancer, particularly breast cancer, cervical cancer, hepatoma, ormelanoma.

Typically, such diagnostic/therapeutic methods involve, first, detectingthe prevalence of cells, such as cancer cells or immune cells, thatexpress the TLR3 polypeptide, and then, if appropriate (e.g., if TLR3expression is detected), administering one or more TLR3 ligands, e.g.,dsRNA, poly I:U, or poly A:U. When the methods are used for thediagnosis or treatment of cancer, e.g., breast cancer, melanoma,cervical cancer, or hepatoma, then the tissue sample will typicallycomprise cancer cells obtained by, e.g., biopsy, and the TLR3 ligandswill be administered to induce the apoptosis of the TLR3-expressingtumor cells. In the case of viral infection or other condition(bacterial or other infection, or other types of cancer), the methodscan involve the detection of TLR3 on immune cells, and the TLR3 ligandsadministered to activate TLR3, leading to an immune reaction (involvinginnate or adaptive immunity) that can target the infected or cancercells for destruction. It will be appreciated that these two methods arenot exclusive, i.e. administering TLR3 ligands in the treatment ofcancer can both activate the immune system via the TLR3 receptor andtrigger apoptosis in TLR3-expressing tumor cells.

As the present antibodies are specific for TLR3, they can also be usedfor other purposes, including purifying TLR3 or TLR3-expressing cells,modulating (e.g. activating or inhibiting) TLR3 receptors in vitro, exvivo, or in vivo, targeting TLR3-expressing cells for destruction invivo, or specifically labeling/binding TLR3 in vivo, ex vivo, or invitro, including for methods such as immunoblotting, FACS analysis, andimmunoprecipitation.

DEFINITIONS

As used herein, “TLR3” ligands refer to any compound that canspecifically bind to and alter the activity of TLR3 in vitro, ex vivo,or in vivo. The compound can be a naturally occurring ligand, e.g.,dsRNA, or a synthetic ligand such as poly-IC or polyAU. The compound canbe any type of molecule, including inorganic or organic compounds orelements, including proteins (such as antibodies), nucleic acids,carbohydrates, lipids, or any other molecular entity. Further, suchcompounds can modulate TLR3 receptors in any way, including activatingor inhibiting, and by any mechanism, including by binding to thereceptor and triggering or shutting off activity in a manner similar toa naturally occurring ligand, or by binding to the receptor and blockingaccess to other ligands. Preferably, the ligand activates the receptor,and as such can be used to induce the apoptosis of TLR3-expressing tumorcells.

As used herein, “paraffin-embedded tissue samples” (or “cells”,“samples”, “slides”, or “tissues”) can refer to cells or tissues takenfrom an organism or from in vitro that have been fixed, embedded inparaffin, sectioned, and transferred to a slide. It will be appreciatedthat fixation and paraffin embedding is a common practice that can varyin many aspects, e.g., with respect to the fixation and embeddingmethods used, with respect to the protocol followed, etc., and that forthe purposes of the present invention any such variant method isencompassed, so long as it involves fixation of the tissue, embedding inparaffin or equivalent material, sectioning and transfer to a slide.

The term “antibody,” as used herein, refers to polyclonal and monoclonalantibodies. Depending on the type of constant domain in the heavychains, antibodies are assigned to one of five major classes: IgA, IgD,IgE, IgG, and IgM. Several of these are further divided into subclassesor isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. An exemplaryimmunoglobulin (antibody) structural unit comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids that is primarily responsible forantigen recognition. The terms variable light chain (V_(L)) and variableheavy chain (V_(H)) refer to these light and heavy chains respectively.The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are termed “alpha,” “delta,” “epsilon,”“gamma” and “mu,” respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. IgG and/or IgM are the preferred classes of antibodiesemployed in this invention, with IgG being particularly preferred,because they are the most common antibodies in the physiologicalsituation and because they are most easily made in a laboratory setting.Preferably the antibody of this invention is a monoclonal antibody.Particularly preferred are humanized, chimeric, human, orotherwise-human-suitable antibodies. “Antibodies” also includes anyfragment or derivative of any of the herein described antibodies.

The term “specifically binds to” means that an antibody can bindpreferably in a competitive binding assay to the binding partner, e.g.TLR3, as assessed using either recombinant forms of the proteins,epitopes therein, or native proteins present on the surface of isolatedtarget cells. Competitive binding assays and other methods fordetermining specific binding are further described below and are wellknown in the art.

When an antibody is said to “compete” or “bind to substantially the sameepitope” as a particular monoclonal antibody (e.g. 40F9), it means thatthe antibody competes with the monoclonal antibody in a binding assayusing either recombinant TLR3 molecules or surface expressed TLR3molecules. For example, if a test antibody reduces the binding of 40F9to a TLR3 polypeptide or TLR3-expressing cell in a binding assay, theantibody is said to “compete” with 40F9.

By “immunogenic fragment,” it is herein meant any polypeptidic orpeptidic fragment that is capable of eliciting an immune response suchas (i) the generation of antibodies binding said fragment and/or bindingany form of the molecule comprising said fragment, including themembrane-bound receptor and mutants derived therefrom, (ii) thestimulation of a T-cell response involving T-cells reacting to thebi-molecular complex comprising any MHC molecule and a peptide derivedfrom said fragment, (iii) the binding of transfected vehicles such asbacteriophages or bacteria expressing genes encoding mammalianimmunoglobulins. Alternatively, an immunogenic fragment also refers toany construction capable of eliciting an immune response as definedabove, such as a peptidic fragment conjugated to a carrier protein bycovalent coupling, a chimeric recombinant polypeptide constructcomprising said peptidic fragment in its amino acid sequence, andspecifically includes cells transfected with a cDNA of which sequencecomprises a portion encoding said fragment.

“Toxic” or “cytotoxic” peptides or small molecules encompass anycompound that can slow down, halt, or reverse the proliferation ofcells, decrease their activity in any detectable way, or directly orindirectly kill them. Preferably, toxic or cytotoxic compounds work bydirectly killing the cells, by provoking apoptosis or otherwise. As usedherein, a toxic “peptide” can include any peptide, polypeptide, orderivative of such, including peptide- or polypeptide-derivatives withunnatural amino acids or modified linkages. A toxic “small molecule” canincludes any toxic compound or element, preferably with a size of lessthan 10 kD, 5 kD, 1 kD, 750 D, 600 D, 500 D, 400 D, 300 D, or smaller.

A “human-suitable” antibody refers to any antibody, derivatizedantibody, or antibody fragment that can be safely used in humans for,e.g. the therapeutic methods described herein. Human-suitable antibodiesinclude all types of humanized, chimeric, or fully human antibodies, orany antibodies in which at least a portion of the antibodies is derivedfrom humans or otherwise modified so as to avoid the immune responsethat is generally provoked when native non-human antibodies are used.

For the purposes of the present invention, a “humanized” or “human”antibody refers to an antibody in which the constant and variableframework region of one or more human immunoglobulins is fused with thebinding region, e.g. the CDR, of an animal immunoglobulin. Suchantibodies are designed to maintain the binding specificity of thenon-human antibody from which the binding regions are derived, but toavoid an immune reaction against the non-human antibody. Such antibodiescan be obtained from transgenic mice or other animals that have been“engineered” to produce specific human antibodies in response toantigenic challenge (see, e.g., Green et al. (1994) Nature Genet 7:13;Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun6:579, the entire teachings of which are herein incorporated byreference). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art (see, e.g., McCafferty et al. (1990)Nature 348:552-553). Human antibodies may also be generated by in vitroactivated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275,which are incorporated in their entirety by reference).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified.

The term “biological sample” as used herein includes but is not limitedto a biological fluid (for example serum, lymph, blood), cell sample, ortissue sample (for example bone marrow or tissue biopsy includingmucosal tissue such as from the gut, gut lamina propria, or lungs).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer. The term“recombinant” when used with reference, e.g., to a cell, or nucleicacid, protein, or vector, indicates that the cell, nucleic acid, proteinor vector, has been modified by the introduction of a heterologousnucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found within thenative (nonrecombinant) form of the cell or express native genes thatare otherwise abnormally expressed, under expressed or not expressed atall.

Within the context of this invention, the term antibody that “binds” acommon determinant designates an antibody that binds said determinantwith specificity and/or affinity.

Producing Anti-TLR3 Antibodies

The antibodies of this invention specifically bind to TLR3 polypeptides,e.g., TLR3 polypeptides on the surface of human cells, particularly inparaffin-embedded tissue sections. The ability of the antibodies tospecifically bind TLR3 polypeptides in paraffin-embedded tissue sectionsmakes them useful for numerous applications, in particular for detectingTLR3 levels or distribution for diagnostic or therapeutic purposes, asdescribed herein. In certain, preferred embodiments, the antibodies areused to determine the presence or level of TLR3 in tumor cells in atissue sample taken from a patient, and, if TLR3 is detected on tumorcells in the tissue sample, TLR3 ligands such as dsRNA, poly-IC orpoly-AU are administered to the patient, thereby inducing apoptosis ofand/or cytokine/chemokine secretion of the TLR3-expressing tumor cells.

In a preferred embodiment, the invention provides an antibody that bindshuman TLR3, and competes for binding to human TLR3 with monoclonalantibody 40F9. Antibody 40F9 is produced by the cell deposited as 40F9.6with the Collection Nationale de Culture de Microorganismes (CNCM),Institut Pasteur, 25 rue de Docteur Roux, F-75724 Paris on 6 Aug. 2008,under the number CNCM 1-4061.

“TLR3”, “TLR3 polypeptide” and “TLR3 receptor”, used interchangeably,are used herein to refer to Toll-Like Receptor 3, a member of theToll-like receptor (TLRs) family. The amino acid sequence of human TLR3is shown in SEQ ID NO:1 (NCBI accession number NP_(—)003256, thedisclosure of which is incorporated herein by reference). The human TLR3mRNA sequence is described in NCBI accession number NM_(—)003265. HumanTLR3 sequences are also described in PCT patent publication no. WO98/50547, the disclosure of which is incorporated herein by reference.

“TLR4”, “TLR4 polypeptide” and “TLR4 receptor”, used interchangeably,are used herein to refer to Toll-Like Receptor 4, a member of theToll-like receptor (TLRs) family. The amino acid sequence of humanprecursor TLR4 including a signal peptide at amino acid residues 1-23 isshown in SEQ ID NO:2 (NCBI accession number NP_(—)612564, the disclosureof which is incorporated herein by reference). Human TLR3 sequences arealso described in THE UniProtKB/Swiss-Prot database and Swissprotaccession number O00206, the disclosure of which is incorporated hereinby reference.

The detection of the binding of the antibody to TLR3 can be performed inany of a number of ways. For example, the antibody can be directlylabeled with a detectable moiety, e.g., a luminescent compound such as afluorescent moiety, or with a radioactive compound, with gold, withbiotin (which allows subsequent, amplified binding to avidin, e.g.,avidin-AP), or with an enzyme such as alkaline phosphatase (AP) orhorseradish peroxidase (HRP). Alternatively, and preferably, the bindingof the antibody to the human TLR3 in the sample is assessed by using asecondary antibody that binds to the primary anti-TLR3 antibody and thatitself is labeled, preferably with an enzyme such as horseradishperoxidase (HRP) or alkaline phosphatase (AP); however, it will beappreciated that the secondary antibodies can be labeled or detectedusing any suitable method. In a preferred embodiment, an amplificationsystem is used to enhance the signal provided by the secondary antibody,for example the EnVision system in which the secondary antibodies arebound to a polymer (e.g., dextran) that is bound to many copies of adetectable compound or enzyme such as HRP or AP (see, e.g., Wiedorn etal. (2001) The Journal of Histochemistry & Cytochemistry, Volume 49(9):1067-1071; Kämmerer et al., (2001) Journal of Histochemistry andCytochemistry, Vol. 49, 623-630; the entire disclosures of which areherein incorporated by reference).

In an advantageous aspect, the invention provides an antibody thatcompetes with monoclonal antibody 40F9 and recognizes, binds to, or hasimmunospecificity for substantially or essentially the same, or thesame, epitope or “epitopic site” on a TLR3 molecule as monoclonalantibody 40F9. In other embodiments, the monoclonal antibody consistsof, or is a derivative or fragment of, antibody 40F9.

It will be appreciated that, while preferred antibodies bind to the sameepitope as antibody 40F9, the present antibodies can recognize and beraised against any part of the TLR3 polypeptide. For example, anyfragment of TLR3, preferably but not exclusively human TLR3, or anycombination of TLR3 fragments, can be used as immunogens to raiseantibodies, and the antibodies of the invention can recognize epitopesat any location within the TLR3 polypeptide, so long as they can do soon paraffin-embedded sections as described herein. Preferably, therecognized epitopes are present on the cell surface, i.e. they areaccessible to antibodies present outside of the cell. Most preferably,the epitope is the epitope specifically recognized by antibody 40F9.Further, antibodies recognizing distinct epitopes within TLR3 can beused in combination, e.g. to bind to TLR3 polypeptides with maximumefficacy and breadth among different individuals or in different tissuesamples.

The antibodies of this invention may be produced by a variety oftechniques known in the art. Typically, they are produced byimmunization of a non-human animal, preferably a mouse, with animmunogen comprising a TLR3 polypeptide, preferably a human TLR3polypeptide. The TLR3 polypeptide may comprise the full length sequenceof a human TLR3 polypeptide, or a fragment or derivative thereof,typically an immunogenic fragment, i.e., a portion of the polypeptidecomprising an epitope exposed on the surface of cells expressing a TLR3polypeptide, preferably the epitope recognized by the 40F9 antibody.Such fragments typically contain at least about 7 consecutive aminoacids of the mature polypeptide sequence, even more preferably at leastabout 10 consecutive amino acids thereof. Fragments typically areessentially derived from the extra-cellular domain of the receptor. In apreferred embodiment, the immunogen comprises a wild-type human TLR3polypeptide in a lipid membrane, typically at the surface of a cell. Ina specific embodiment, the immunogen comprises intact cells,particularly intact human cells, optionally treated or lysed. In anotherpreferred embodiment, the polypeptide is a recombinant TLR3 polypeptide.

The step of immunizing a non-human mammal with an antigen may be carriedout in any manner well known in the art for stimulating the productionof antibodies in a mouse (see, for example, E. Harlow and D. Lane,Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988), the entire disclosure of which isherein incorporated by reference). The immunogen is suspended ordissolved in a buffer, optionally with an adjuvant, such as complete orincomplete Freund's adjuvant. Methods for determining the amount ofimmunogen, types of buffers and amounts of adjuvant are well known tothose of skill in the art and are not limiting in any way on the presentinvention. These parameters may be different for different immunogens,but are easily elucidated.

Similarly, the location and frequency of immunization sufficient tostimulate the production of antibodies is also well known in the art. Ina typical immunization protocol, the non-human animals are injectedintraperitoneally with antigen on day 1 and again about a week later.This is followed by recall injections of the antigen around day 20,optionally with an adjuvant such as incomplete Freund's adjuvant. Therecall injections are performed intravenously and may be repeated forseveral consecutive days. This is followed by a booster injection at day40, either intravenously or intraperitoneally, typically withoutadjuvant. This protocol results in the production of antigen-specificantibody-producing B cells after about 40 days. Other protocols may alsobe used as long as they result in the production of B cells expressingan antibody directed to the antigen used in immunization.

For polyclonal antibody preparation, serum is obtained from an immunizednon-human animal and the antibodies present therein isolated bywell-known techniques. The serum may be affinity purified using any ofthe immunogens set forth above linked to a solid support so as to obtainantibodies that react with TLR3 polypeptides.

In an alternate embodiment, lymphocytes from a non-immunized non-humanmammal are isolated, grown in vitro, and then exposed to the immunogenin cell culture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out.

For preferred monoclonal antibodies, the next step is the isolation ofsplenocytes from the immunized non-human mammal and the subsequentfusion of those splenocytes with an immortalized cell in order to forman antibody-producing hybridoma. The isolation of splenocytes from anon-human mammal is well-known in the art and typically involvesremoving the spleen from an anesthetized non-human mammal, cutting itinto small pieces and squeezing the splenocytes from the splenic capsulethrough a nylon mesh of a cell strainer into an appropriate buffer so asto produce a single cell suspension. The cells are washed, centrifugedand resuspended in a buffer that lyses any red blood cells. The solutionis again centrifuged and remaining lymphocytes in the pellet are finallyresuspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes canbe fused to an immortal cell line. This is typically a mouse myelomacell line, although many other immortal cell lines useful for creatinghybridomas are known in the art. Preferred murine myeloma lines include,but are not limited to, those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, U.S.A., X63 Ag8653 and SP-2 cells available from the AmericanType Culture Collection, Rockville, Md. U.S.A. The fusion is effectedusing polyethylene glycol or the like. The resulting hybridomas are thengrown in selective media that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Hybridomas are typically grown on a feeder layer of macrophages. Themacrophages are preferably from littermates of the non-human mammal usedto isolate splenocytes and are typically primed with incomplete Freund'sadjuvant or the like several days before plating the hybridomas. Fusionmethods are described in Goding, “Monoclonal Antibodies: Principles andPractice,” pp. 59-103 (Academic Press, 1986), the disclosure of which isherein incorporated by reference.

The cells are allowed to grow in the selection media for sufficient timefor colony formation and antibody production. This is usually betweenabout 7 and about 14 days.

The hybridoma colonies are then assayed for the production of antibodiesthat specifically bind to TLR3 polypeptide gene products, preferably theepitope specifically recognized by antibody 40F9. The assay is typicallya colorimetric ELISA-type assay, although any assay may be employed thatcan be adapted to the wells that the hybridomas are grown in. Otherassays include radioimmunoassays or fluorescence activated cell sorting.The wells positive for the desired antibody production are examined todetermine if one or more distinct colonies are present. If more than onecolony is present, the cells may be re-cloned and grown to ensure thatonly a single cell has given rise to the colony producing the desiredantibody. Typically, the antibodies will also be tested for the abilityto bind to TLR3 polypeptides, e.g., TLR3-expressing cells, inparaffin-embedded tissue sections, as described below.

Hybridomas that are confirmed to produce a monoclonal antibody of thisinvention can be grown up in larger amounts in an appropriate medium,such as DMEM or RPMI-1640. Alternatively, the hybridoma cells can begrown in vivo as ascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, thegrowth media containing monoclonal antibody (or the ascites fluid) isseparated away from the cells and the monoclonal antibody presenttherein is purified. Purification is typically achieved by gelelectrophoresis, dialysis, chromatography using protein A or proteinG-Sepharose, or an anti-mouse Ig linked to a solid support such asagarose or Sepharose beads (all described, for example, in the AntibodyPurification Handbook, Biosciences, publication No. 18-1037-46, EditionAC, the disclosure of which is hereby incorporated by reference). Thebound antibody is typically eluted from protein A/protein G columns byusing low pH buffers (glycine or acetate buffers of pH 3.0 or less) withimmediate neutralization of antibody-containing fractions. Thesefractions are pooled, dialyzed, and concentrated as needed.

Positive wells with a single apparent colony are typically re-cloned andre-assayed to insure only one monoclonal antibody is being detected andproduced.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in (Ward et al. Nature,341 (1989) p. 544, the entire disclosure of which is herein incorporatedby reference).

The identification of one or more antibodies that bind(s) to TLR3,particularly substantially or essentially the same epitope as monoclonalantibody 40F9, can be readily determined using any one of a variety ofimmunological screening assays in which antibody competition can beassessed. Many such assays are routinely practiced and are well known inthe art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997, whichis specifically incorporated herein by reference). It will be understoodthat actually determining the epitope to which an antibody describedherein binds is not in any way required to identify an antibody thatbinds to the same or substantially the same epitope as the monoclonalantibody described herein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control (40F9, forexample) and test antibodies are admixed (or pre-adsorbed) and appliedto a sample containing TLR3 polypeptides. Protocols based upon westernblotting and the use of BIACORE analysis are suitable for use in suchsimple competition studies.

In certain embodiments, one pre-mixes the control antibodies (40F9, forexample) with varying amounts of the test antibodies (e.g., about 1:10or about 1:100) for a period of time prior to applying to the TLR3antigen sample. In other embodiments, the control and varying amounts oftest antibodies can simply be admixed during exposure to the TLR3antigen sample. As long as one can distinguish bound from freeantibodies (e.g., by using separation or washing techniques to eliminateunbound antibodies) and 40F9 from the test antibodies (e.g., by usingspecies-specific or isotype-specific secondary antibodies or byspecifically labeling 40F9 with a detectable label) one can determine ifthe test antibodies reduce the binding of 40F9 to the antigens,indicating that the test antibody recognizes substantially the sameepitope as 40F9. The binding of the (labeled) control antibodies in theabsence of a completely irrelevant antibody can serve as the controlhigh value. The control low value can be obtained by incubating thelabeled (40F9) antibodies with unlabelled antibodies of exactly the sametype (40F9), where competition would occur and reduce binding of thelabeled antibodies. In a test assay, a significant reduction in labeledantibody reactivity in the presence of a test antibody is indicative ofa test antibody that recognizes substantially the same epitope, i.e.,one that “cross-reacts” with the labeled (40F9) antibody. Any testantibody that reduces the binding of 40F9 to TLR3 antigens by at leastabout 50%, such as at least about 60%, or more preferably at least about70% (e.g., about 65-100%), at any ratio of 40F9:test antibody betweenabout 1:10 and about 1:100 is considered to be an antibody that binds tosubstantially the same epitope or determinant as 40F9. Preferably, suchtest antibody will reduce the binding of 40F9 to the TLR3 antigen by atleast about 90% (e.g., about 95%).

Competition can also be assessed by, for example, a flow cytometry test.In such a test, cells bearing a given TLR3 polypeptide can be incubatedfirst with 40F9, for example, and then with the test antibody labeledwith a fluorochrome or biotin. The antibody is said to compete with 40F9if the binding obtained upon preincubation with a saturating amount of40F9 is about 80%, preferably about 50%, about 40% or less (e.g., about30%) of the binding (as measured by mean of fluorescence) obtained bythe antibody without preincubation with 40F9. Alternatively, an antibodyis said to compete with 40F9 if the binding obtained with a labeled 40F9antibody (by a fluorochrome or biotin) on cells preincubated with asaturating amount of test antibody is about 80%, preferably about 50%,about 40%, or less (e.g., about 30%) of the binding obtained withoutpreincubation with the antibody.

A simple competition assay in which a test antibody is pre-adsorbed andapplied at saturating concentration to a surface onto which a TLR3antigen is immobilized may also be employed. The surface in the simplecompetition assay is preferably a BIACORE chip (or other media suitablefor surface plasmon resonance analysis). The control antibody (e.g.,40F9) is then brought into contact with the surface at a TLR3-saturatingconcentration and the TLR3 and surface binding of the control antibodyis measured. This binding of the control antibody is compared with thebinding of the control antibody to the TLR3-containing surface in theabsence of test antibody. In a test assay, a significant reduction inbinding of the TLR3-containing surface by the control antibody in thepresence of a test antibody indicates that the test antibody recognizessubstantially the same epitope as the control antibody such that thetest antibody “cross-reacts” with the control antibody. Any testantibody that reduces the binding of control (such as 40F9) antibody toa TLR3 antigen by at least about 30% or more, preferably about 40%, canbe considered to be an antibody that binds to substantially the sameepitope or determinant as a control (e.g., 40F9). Preferably, such atest antibody will reduce the binding of the control antibody (e.g.,40F9) to the TLR3 antigen by at least about 50% (e.g., at least about60%, at least about 70%, or more). It will be appreciated that the orderof control and test antibodies can be reversed: that is, the controlantibody can be first bound to the surface and the test antibody isbrought into contact with the surface thereafter in a competition assay.Preferably, the antibody having higher affinity for the TLR3 antigen isbound to the surface first, as it will be expected that the decrease inbinding seen for the second antibody (assuming the antibodies arecross-reacting) will be of greater magnitude. Further examples of suchassays are provided in, e.g., Saunal H. and al (1995) J. Immunol.Methods 183: 33-41, the disclosure of which is incorporated herein byreference.

Preferably, monoclonal antibodies that recognize a TLR3 epitope willreact with an epitope that is present on a substantial percentage of oreven all TLR3-expressing cells, e.g., dendritic cells, CD8+ T cells,cells of the skin, cerebellum, breast, lung, esophagus, stomach, ileum,jejunum, duodenum, colon, liver, pancreas, testis, spleen, thymus, ortonsil, but will not significantly react with other cells, i.e., immuneor non-immune cells that do not express TLR3.

In preferred embodiments, the antibodies will bind to TLR3-expressingcells from an individual or individuals with a disorder associated withTLR3 expression e.g., tumors such as breast cancer, cervical cancer,hepatoma, melanoma, i.e., an individual that is a candidate fortreatment with one of the herein-described methods (e.g., using a TLR3ligand). Accordingly, once an antibody that specifically recognizes TLR3on cells, preferably human cells, it can be tested for its ability tobind to TLR3-expressing cells taken from a patient with a condition suchas breast cancer, melanoma, hepatoma, cervical cancer, or cancer of thelung, esophagus, stomach, or larynx.

In one embodiment of any of the methods of the present invention, tumorcells, e.g., melanoma cells, are first treated with a cytokine such as atype-1 interferon or with a TLR3 ligand prior to antibody binding, assuch treatments can enhance TLR3 expression and thus facilitate antibodybinding under some conditions.

In one embodiment, the antibodies of the invention are validated in animmunoassay to test their ability to bind to TLR3-expressing cells, e.g.tonsil cells. Preferably, the validation is performed by assessing theability of the antibody to stain TLR3-expressing cells in aparaffin-embedded tissue section. For example, tonsil tissue samples aretaken from a plurality of patients, and the ability of a given antibodyto stain cells within the tissue is then assessed using standard methodswell known to those in the art. Antibodies that are found to bind to asubstantial proportion (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% or more)of tissue samples known to contain TLR3-expressing cells, e.g. tonsilcells or certain tumors, from a significant percentage of individuals orpatients (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) are suitablefor use in the present invention, both for diagnostic purposes todetermine the presence or level of TLR3 cells in a patient or for use inthe herein-described therapeutic methods, e.g., to detectTLR3-expressing tumor cells prior to the administration of TLR3 ligands.To assess the binding of the antibodies to the cells, the antibodies caneither be directly or indirectly labeled. When indirectly labeled, asecondary, labeled antibody is typically added. Such methods are wellknown to those of skill in the art and are described further elsewhereherein.

While described in the context of 40F9 for the purposes ofexemplification, it will be appreciated that the herein-describedimmunological screening assays and other assays can also be used toidentify antibodies that compete with other anti-TLR3 antibodies, solong as they also bind to TLR3 in paraffin-embedded tissue samples.

Determination of whether an antibody binds within one of the epitoperegions defined above can be carried out in ways known to the personskilled in the art. As one example of such mapping/characterizationmethods, an epitope region for an anti-TLR3 antibody may be determinedby epitope “foot-printing” using chemical modification of the exposedamines/carboxyls in the TLR3 protein. One specific example of such afoot-printing technique is the use of HXMS (hydrogen-deuterium exchangedetected by mass spectrometry) wherein a hydrogen/deuterium exchange ofreceptor and ligand protein amide protons, binding, and back exchangeoccurs, wherein the backbone amide groups participating in proteinbinding are protected from back exchange and therefore will remaindeuterated. Relevant regions can be identified at this point by pepticproteolysis, fast microbore high-performance liquid chromatographyseparation, and/or electrospray ionization mass spectrometry. See, e.g.,Ehring H, Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999)Engen, J. R. and Smith, D. L. (2001) Anal. Chem. 73, 256A-265A. Anotherexample of a suitable epitope identification technique is nuclearmagnetic resonance (NMR) epitope mapping, where typically the positionof the signals in two-dimensional NMR spectra of the free antigen andthe antigen complexed with the antigen binding peptide, such as anantibody, are compared. The antigen typically is selectivelyisotopically labeled with 15N so that only signals corresponding to theantigen and no signals from the antigen binding peptide are seen in theNMR-spectrum. Antigen signals originating from amino acids involved inthe interaction with the antigen binding peptide typically will shiftposition in the spectrum of the complex compared to the spectrum of thefree antigen, and the amino acids involved in the binding can beidentified that way. See, e.g., Ernst Schering Res Found Workshop. 2004;(44): 149-67; Huang et al, Journal of Molecular Biology, Vol. 281 (1)pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 June; 9 (3):516-24.

Epitope mapping/characterization also can be performed using massspectrometry methods. See, e.g., Downward, J Mass Spectrom. 2000 April;35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9):1792-801. Protease digestion techniques also can be useful in thecontext of epitope mapping and identification. Antigenicdeterminant-relevant regions/sequences can be determined by proteasedigestion, e.g. by using trypsin in a ratio of about 1:50 to TLR3 or o/ndigestion at and pH 7-8, followed by mass spectrometry (MS) analysis forpeptide identification. The peptides protected from trypsin cleavage bythe anti-TLR3 binder can subsequently be identified by comparison ofsamples subjected to trypsin digestion and samples incubated withantibody and then subjected to digestion by e.g. trypsin (therebyrevealing a footprint for the binder). Other enzymes like chymotrypsin,pepsin, etc., also or alternatively can be used in similar epitopecharacterization methods. Moreover, enzymatic digestion can provide aquick method for analyzing whether a potential antigenic determinantsequence is within a region of the TLR3 polypeptide that is not surfaceexposed and, accordingly, most likely not relevant in terms ofimmunogenicity/antigenicity. See, e.g., Manca, Ann Ist Super Sanita.1991; 27: 15-9 for a discussion of similar techniques.

Once antibodies that are capable of binding to TLR3 in paraffin sectionsare identified, they will also typically be assessed, using standardmethods including those described herein, for their ability to bind toother polypeptides, including unrelated polypeptides and other TLRfamily members (e.g., human TLR1, 2, or 4-10). Ideally, the antibodiesonly bind with substantial affinity to TLR3, e.g., human TLR3, and donot bind at a significant level to unrelated polypeptides or to otherTLR family members (e.g., TLR2 or TLR4). However, it will be appreciatedthat, as long as the affinity for TLR3 is substantially greater (e.g.,5×, 10×, 50×, 100×, 500×, 1000×, 10,000×, or more) than it is for otherTLR family members (or other, unrelated polypeptides), then theantibodies are suitable for use in the present methods. The preferredantibodies of the invention do not substantially bind to human TLR4,e.g. do not stain samples (e.g. paraffin-embedded samples) which expressTLR4 and do not express TLR3. Human TLR4, a receptor forlipopolysaccharide (LPS) is described in OMIM Accession number 603030,and mRNA and amino acid sequences are provided in Genbank Accessionnumber U88880 and AAC34135, as well as in Rock et al. (1998) P.N.A.S.USA 95(2): 588-593, the disclosures of which are incorporated herein byreference.

The binding of the antibodies to TLR3-expressing cells can also beassessed in non-human primates, e.g. cynomolgus monkeys, or othermammals such as mice. The invention therefore provides an antibody, aswell as fragments and derivatives thereof, wherein said antibody,fragment or derivative specifically binds to TLR3 polypeptides at thesurface of human cells present in a paraffin-embedded tissue section,and which furthermore binds to TLR3 expressing cells from non-humanprimates, e.g., cynomolgus monkeys. In certain embodiments, thenon-human primate is a model for a TLR3-associated condition such asbreast cancer or melanoma.

Upon immunization and production of antibodies in a vertebrate or cell,particular selection steps may be performed to isolate antibodies asclaimed. In this regard, in a specific embodiment, the invention alsorelates to methods of producing such antibodies, comprising: (a)immunizing a non-human mammal with an immunogen comprising a TLR3polypeptide; and (b) preparing antibodies from said immunized animal;and (c) selecting antibodies from step (b) that are capable of bindingsaid TLR3 polypeptide in a paraffin-embedded tissue sample. In oneembodiment, the method further comprises a step (d), selectingantibodies from (b) that are capable of competing for binding to TLR3with antibody 40F9.

In preferred embodiments, the antibodies prepared according to thepresent methods are monoclonal antibodies. In preferred embodiments, thenon-human animal used to produce antibodies according to the methods ofthe invention is a mammal, such as a rodent, bovine, porcine, horse,rabbit, goat, or sheep.

According to an alternate embodiment, the DNA encoding an antibody thatbinds an epitope present on TLR3 polypeptides is isolated from thehybridoma of this invention and placed in an appropriate expressionvector for transfection into an appropriate host. The host is then usedfor the recombinant production of the antibody, or variants thereof,such as a humanized version of that monoclonal antibody, activefragments of the antibody, chimeric antibodies comprising the antigenrecognition portion of the antibody, or versions comprising a detectablemoiety.

DNA encoding the monoclonal antibodies of the invention, e.g., antibody40F9, can be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofmurine antibodies). Once isolated, the DNA can be placed into expressionvectors, which are then transfected into host cells such as E. colicells, simian COS cells, Chinese hamster ovary (CHO) cells, or myelomacells that do not otherwise produce immunoglobulin protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells. Asdescribed elsewhere in the present specification, such DNA sequences canbe modified for any of a large number of purposes, e.g., for humanizingantibodies, producing fragments or derivatives, or for modifying thesequence of the antibody, e.g., in the antigen binding site in order tooptimize the binding specificity of the antibody.

Recombinant expression in bacteria of DNA encoding the antibody is wellknown in the art (see, for example, Skerra et al., Curr. Opinion inImmunol., 5, pp. 256 (1993); and Pluckthun, Immunol. 130, pp. 151(1992).

Antibody 40F9

In any of the embodiments herein, antibody 40F9 can be characterized byits amino acid sequence and/or nucleic acid sequence encoding it. Theamino acid sequences of variable regions of the heavy and light chainsfor antibody 40F9 are listed in SEQ ID NOS:3 and 4, respectively. SEQ IDNO:3 shows the 40F9 light chain variable region (40F9VL) which wasobtained as a potential rearrangement of VKappa bd2 (amino acidpositions 1-102)/JK2 (amino acid positions 102-113)/Ckappa SEQ ID NO: 4shows the 40F9 heavy chain variable region (40F9VK) which was obtainedas a potential rearrangement of VH9-10 (amino acid positions1-98)/DSP2.2 (amino acid positions 99-102)/JH2 (amino acid positions103-113)/Cgamma1. In a specific embodiment, the antibody bindsessentially the same epitope or determinant as one of monoclonalantibodies 40F9. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 40F9. According to anotherpreferred embodiment, the monoclonal antibody comprises the three CDRsof the variable heavy chain region of 40F9 (CDR1=amino acids 24 to 39 ofSEQ ID NO:3; CDR2=amino acids 55 to 61 of SEQ ID NO:3; CDR3=amino acids94-102 of SEQ ID NO:3). Also provided is a monoclonal antibody thatcomprises the variable heavy chain region of 40F9 (40F9VH; SEQ ID NO:3).According to another preferred embodiment, the monoclonal antibodycomprises the three CDRs of the variable light chain region of 40F9(CDR1=amino acids 27 to 35 of SEQ ID NO:4; CDR2=amino acids 52 to 66 ofSEQ ID NO:4; CDR3=amino acids 99-103 of SEQ ID NO:4). Also provided is amonoclonal antibody that comprises the variable light chain region of40F9 (40F9VK; SEQ ID NO:4).

In another preferred embodiment, the antibody comprises a light chaincomprising one, two or three of the CDRs of the variable light chainregion of 40F9. In one embodiment, the CDRs of the variable light chainregion comprise an amino acid sequence selected from the groupconsisting of: KSSQSLLDSDGKTYLN (SEQ ID NO:5; CDR1); LVSKLDS (SEQ IDNO:6; CDR2); and WQGIHLPYT (SEQ ID NO:7; CDR3), or any sequence of 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous amino acidsthereof (to the extent such sequence is consistent with the length ofthe SEQ ID), or any sequence which is at least 50%, 60%, 70%, 80% or 90%identical thereto over the length of the aforementioned CDR sequence. Inanother preferred embodiment, the antibody comprises a light chaincomprising one, two or three of the CDRs of the variable heavy chainregion of 40F9. In one embodiment, the CDRs of the variable heavy chainregion comprise an amino acid sequence selected from the groupconsisting of: YTFTNYGMN (SEQ ID NO:8; CDR1); NANTGEPTYAEEFKG (SEQ IDNO:9; CDR2); and DYDY (SEQ ID NO:10; CDR3), or any sequence of 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous amino acids thereof (tothe extent such sequence is consistent with the length of the SEQ ID),or any sequence which is at least 50%, 60%, 70%, 80% or 90% identicalthereto over the length of the sequence of the aforementioned CDRsequence. CDR1 of the heavy chain in SEQ ID NO:8 is defined according tothe Kabat definition; however heavy chain CDR1 can alternatively bedefined according to the Chothia definition as KASGYTFTNYGMN (SEQ IDNO:11; CDR1). It will therefore be appreciated that in any of theembodiments herein, the heavy chain CDR1 described in SEQ ID NO:11 maybe substituted for the CDR1 described in SEQ ID NO:8. Optionally any oneor more of said light or heavy chain CDRs may contain one, two, three,four or five amino acid modifications (e.g. substitutions, insertions ordeletions). In another preferred embodiment the antibody is 40F9.

Fragments and Derivatives of the Present Monoclonal Antibodies

Fragments and derivatives of antibodies of this invention (which areencompassed by the term “antibody” or “antibodies” as used in thisapplication, unless otherwise stated or clearly contradicted bycontext), preferably a 40F9-like antibody, can be produced by techniquesthat are known in the art. “Fragments” comprise a portion of the intactantibody, generally the antigen binding site or variable region.Examples of antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)₂, andFv fragments; diabodies; any antibody fragment that is a polypeptidehaving a primary structure consisting of one uninterrupted sequence ofcontiguous amino acid residues (referred to herein as a “single-chainantibody fragment” or “single chain polypeptide”), including withoutlimitation (1) single-chain Fv molecules (2) single chain polypeptidescontaining only one light chain variable domain, or a fragment thereofthat contains the three CDRs of the light chain variable domain, withoutan associated heavy chain moiety and (3) single chain polypeptidescontaining only one heavy chain variable region, or a fragment thereofcontaining the three CDRs of the heavy chain variable region, without anassociated light chain moiety; and multispecific antibodies formed fromantibody fragments.

Fragments of the present antibodies can be obtained using standardmethods. For instance, Fab or F(ab′)₂ fragments may be produced byprotease digestion of the isolated antibodies, according to conventionaltechniques. It will be appreciated that immunoreactive fragments can bemodified using known methods, for example to slow clearance in vivo andobtain a more desirable pharmacokinetic profile the fragment may bemodified with polyethylene glycol (PEG). Methods for coupling andsite-specifically conjugating PEG to a Fab′ fragment are described in,for example, Leong et al, 16 (3): 106-119 (2001) and Delgado et al, Br.J. Cancer 73 (2): 175-182 (1996), the disclosures of which areincorporated herein by reference.

Alternatively, the DNA of a hybridoma producing an antibody of theinvention, preferably a 40F9-like antibody, may be modified so as toencode a fragment of the invention. The modified DNA is then insertedinto an expression vector and used to transform or transfect anappropriate cell, which then expresses the desired fragment.

In certain embodiments, the DNA of a hybridoma producing an antibody ofthis invention, preferably a 40F9-like antibody, can be modified priorto insertion into an expression vector, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous non-human sequences (e.g., Morrison et al., PNASpp. 6851 (1984)), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of the original antibody.Typically, such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention.

Thus, according to another embodiment, the antibody of this invention,preferably a 40F9-like antibody, is humanized. “Humanized” forms ofantibodies according to this invention are specific chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂, or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from the murine immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of theoriginal antibody (donor antibody) while maintaining the desiredspecificity, affinity, and capacity of the original antibody.

In some instances, Fv framework residues of the human immunoglobulin maybe replaced by corresponding non-human residues. Furthermore, humanizedantibodies can comprise residues that are not found in either therecipient antibody or in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof the original antibody and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature, 321, pp.522 (1986); Reichmann et Nature, 332, pp. 323 (1988); Presta, Curr. Op.Struct. Biol., 2, pp. 593 (1992); Verhoeyen et Science, 239, pp. 1534;and U.S. Pat. No. 4,816,567, the entire disclosures of which are hereinincorporated by reference.) Methods for humanizing the antibodies ofthis invention are well known in the art.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of an antibody of this invention is screenedagainst the entire library of known human variable-domain sequences. Thehuman sequence which is closest to that of the mouse is then accepted asthe human framework (FR) for the humanized antibody (Sims et al., J.Immunol. 151, pp. 2296 (1993); Chothia and Lesk, J. Mol. 196, pp. 901).Another method uses a particular framework from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework can be used for several different humanizedantibodies (Carter et al., PNAS 89, pp. 4285 (1992); Presta et J.Immunol., 51, pp. 1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for TLR3 receptors and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional structures ofselected candidate immunoglobulin sequences. Inspection of thesedisplays permits analysis of the likely role of the residues in thefunctioning of the candidate immunoglobulin sequence, i.e., the analysisof residues that influence the ability of the candidate immunoglobulinto bind its antigen. In this way, FR residues can be selected andcombined from the consensus and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen (s), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding.

Another method of making “humanized” monoclonal antibodies is to use aXenoMouse (Abgenix, Fremont, Calif.) as the mouse used for immunization.A XenoMouse is a murine host according to this invention that has hadits immunoglobulin genes replaced by functional human immunoglobulingenes. Thus, antibodies produced by this mouse or in hybridomas madefrom the B cells of this mouse, are already humanized. The XenoMouse isdescribed in U.S. Pat. No. 6,162,963, which is herein incorporated inits entirety by reference.

Human antibodies may also be produced according to various othertechniques, such as by using, for immunization, other transgenic animalsthat have been engineered to express a human antibody repertoire(Jakobovitz et Nature 362 (1993) 255), or by selection of antibodyrepertoires using phage display methods. Such techniques are known tothe skilled person and can be implemented starting from monoclonalantibodies as disclosed in the present application.

The antibodies of the present invention, preferably a 40F9-likeantibody, may also be derivatized to “chimeric” antibodies(immunoglobulins) in which a portion of the heavy/light chain(s) isidentical with or homologous to corresponding sequences in the originalantibody, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity and binding specificity (Cabilly et al., supra;Morrison et al., Proc. Natl. Acad. Sci. U.S.A., pp. 6851 (1984)).

While the present antibodies all have the ability to bind to TLR3 inparaffin embedded or equivalent tissue samples, they can also be usedfor other purposes such as to label TLR3 in vitro, ex vivo, or in vivo,to purify TLR3-expressing cells, or to target TLR3-expressing cells forkilling, e.g., in vivo. Accordingly, other derivatives of the presentantibodies that fall within the scope of this invention includefunctionalized antibodies, i.e., antibodies that are conjugated orcovalently bound to a toxin, such as ricin, diphtheria toxin, abrin, orPseudomonas exotoxin; to a detectable moiety, such as a fluorescentmoiety, a radioisotope, or an imaging agent; or to a solid support, suchas agarose beads or the like. Methods for conjugation or covalentbonding of these other agents to antibodies are well known in the art.Conjugation to a toxin is useful for targeted killing cells displayingTLR3 receptors on its cell surface, e.g. certain tumor cells such asmelanomas or those of the breast, lung, esophagus, stomach, larynx,kidney, or cervix. In such embodiments, typically a biopsy will beperformed initially to assess whether the tumor cells express TLR3,e.g., using the detection methods described herein. If TLR3 is indeeddetected on the surface of the tumor cells, then, together with or as analternative to the administration of TLR3 ligands as described elsewhereherein, cytotoxic antibodies can be administered. Once the cytotoxicantibody of the invention binds to the cell surface of TLR3-expressingcells, e.g., TLR3-expressing tumor cells, it is internalized and thetoxin is released inside of the cell, selectively killing that cell.

Conjugation to a detectable moiety is useful, inter alia, when anantibody of the invention is used for diagnostic purposes. Such purposesinclude, but are not limited to, assaying biological samples, e.g., ablood sample or tissue biopsy, for the presence of TLR3-expressingcells, and detecting the presence, level, or activity of TLR3-expressingcells in an individual. Such assay and detection methods can be used inthe diagnostic/therapeutic methods of the invention, e.g., involvingdetecting TLR3 expression in tumor cells of a patient and subsequentlyadministering a TLR3 ligand, e.g., dsRNA, polyIC, polyAU, to the patientto induce apoptosis of the TLR3-expressing cells.

In certain embodiments, the present antibodies are used to purifyTLR3-expressing cells from a biological sample. Biological samples canbe obtained from a patient, e.g. for diagnostic or ex vivo therapeuticpurposes, or from individuals or non-human primates to obtain a sourceof such cells for research purposes.

In one such embodiment, labeled antibodies of the invention can be usedin FACS sorting to purify or isolate TLR3-expressing cells from abiological sample. Alternatively, in some embodiments conjugation of anantibody of this invention to a solid support can be useful as a toolfor affinity purification of cells bearing a TLR3 receptor on their cellsurface from a biological sample, such as a blood sample or cells from atissue biopsy from an individual. This method of purification is anotheralternate embodiment of the present invention, as is the resultingpurified population of cells.

Regardless of the method used to isolate or purify the TLR3-expressingcells, the ability to do so is useful for numerous purposes, e.g. todiagnose a TLR3-associated disorder by assessing the number or activityof TLR3-expressing cells, e.g., prior to administration of TLR3 ligandsas described herein. Also, purifying or isolating cells can be performedto evaluate the sensitivity of the TLR3-expressing cells undergoingapoptosis in the presence of TLR3 ligands. Further, purifiedTLR3-expressing cells are useful in a research context, e.g., to bettercharacterize the cells and their various properties and behaviors, aswell as to identify compounds or methods that can be used to modulatetheir behavior, activity, survival, or proliferation.

Preparation and Staining of Paraffin-Embedded Tissue Sections

The present antibodies have the particular property of being able toefficiently and specifically bind to TLR3 polypeptides present in fixedtissue or cell samples. Without being bound by the following theory, itis believed that fixation, e.g., formalin fixation, may destroy manyepitopes present on the polypeptide, eliminating the ability of manyantibodies to specifically bind to the polypeptide; alternatively, thefixation/embedding procedure may expose or render particularlyaccessible particular epitopes that are not necessarily readily presentfor antibody binding by other methods, e.g., western blot, FACS, etc.The present antibodies are not only able to bind such epitopes with highaffinity, but preferably also do so with high specificity, e.g., they donot substantially bind to other polypeptides, e.g., unrelated proteinsor other TLR family members such as TLR4.

Various methods of preparing and using such tissue preparations are wellknown in the art, and any suitable method or type of preparation can beused. It will be appreciated, for example, that the present antibodiescan be used with any fixed cell or tissue preparation, and that they arenot limited by the particular fixation or embedding method used. Forexample, while the most common fixation procedure involves formalin(e.g., 10%), alternative methods such as paraformaldehyde (PFA), Bouinsolution (formalin/picric acid), alcohol, zinc-based solutions (for oneexample, see, e.g., Lykidis et al., (2007) Nucleic Acids Research, 2007,1-10, the entire disclosure of which is herein incorporated in itsentirety), and others (see, e.g., the HOPE method, Pathology Researchand Practice, Volume 197, Number 12, December 2001, pp. 823-826(4), theentire disclosure of which is herein incorporated by reference).Similarly, while paraffin is preferred, other materials can be used forembedding as well, e.g., polyester wax, polyethylene glycol basedformulas, glycol methacrylates, JB-4 plastics, and others. For review ofmethods for preparing and using tissue preparations, see, e.g.,Gillespie et al., (2002) Am J. Pathol. 2002 February; 160(2): 449-457;Fischer et al. CSH Protocols; 2008; Renshaw (2007),Immunohistochemistry: Methods Express Series; Bancroft (2007) Theory andPractice of Histological Techniques; and PCT patent publication no.WO06074392; the entire disclosures of which are herein incorporated byreference).

Generally, the tissue (or cells) to be examined is obtained by, e.g.,biopsy from a tumor tissue (e.g., breast tumor, melanoma) or from ahealthy tissue, and sections (e.g., 3 mm thick or less) and fixed usingformalin or an equivalent fixation method (see supra). The time offixation depends on the application and is not critical for the purposesof the present invention, but can range from several hours to 24 or morehours. Following fixation, the tissue is embedded in paraffin (orequivalent material), and very thin sections (e.g., 5 microns) are cutin a microtome and then mounted onto, preferably coated, slides. Theslides are then dried, e.g., air dried.

Fixed and embedded tissue sections on slides can be dried and storedindefinitely. For immunohistochemistry, the slides are deparaffinizedand then rehydrated. For example, they are subjected to a series ofwashes with, initially, xylene, and then xylene with ethanol, and thenwith decreasing percentages of ethanol in water.

Before antibody staining, the tissues can be subjected to an antigenretrieval step, e.g., enzymatic or heat-based, in order to break methanebridges that form during fixation and which can mask epitopes. In apreferred embodiment, a treatment in boiling 10 mM citrate buffer, pH 6,is used.

Once the slides have been rehydrated and antigen retrieval has beenideally performed, they can be incubated with the primary antibody.First, the slides are washed with, e.g., TBS, and then, following ablocking step with, e.g., serum/BSA, the antibody can be applied. Theconcentration of the antibody will depend on its form (e.g., purified),its affinity, the tissue sample used, but a suitable concentration is,e.g., 1-10 μg/ml. In one embodiment, the concentration used is 10 μg/ml.The time of incubation can vary as well, but an overnight incubation istypically suitable. Following a post-antibody washing step in, e.g.,TBS, the slides are then processed for detection of antibody binding.

The detection method used will depend on the antibody, tissue, etc.used, and can involve detection of a luminescent or otherwise visible ordetectable moiety conjugated to the primary antibody, or through the useof detectable secondary antibodies. Methods of antibody detection arewell known in the art and are taught, e.g., in Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,1st edition (Dec. 1, 1988); Fischer et al. CSH Protocols; 2008; Renshaw(2007), Immunohistochemistry: Methods Express Series; Bancroft (2007)Theory and Practice of Histological Techniques; PCT patent publicationno. WO06074392; the entire disclosure of each of which is hereinincorporated in its entirety.

In a preferred embodiment, the binding of the primary antibody isdetected by binding a labeled secondary antibody, preferably a secondaryantibody covalently linked to an enzyme such as HRP or AP. In aparticularly preferred embodiment, the signal generated by binding ofthe secondary antibody is amplified using any of a number of methods foramplification of antibody detection. For example, the EnVision methodcan be used, (see, e.g., U.S. Pat. No. 5,543,332 and European Patent no.594,772; Kämmerer et al., (2001) Journal of Histochemistry andCytochemistry, Vol. 49, 623-630; Wiedorn et al. (2001) The Journal ofHistochemistry & Cytochemistry, Volume 49(9): 1067-1071; the entiredisclosures of which are herein incorporated by reference), in which thesecondary antibodies are linked to a polymer (e.g., dextran) that isitself linked to many copies of AP or HRP.

Assessing the Ability of Antibodies to Modulate TLR3-Expressing CellActivity

In certain embodiments, the antibodies of this invention are able tomodulate, e.g., activate or inhibit, the activity or behavior ofTLR3-expressing cells, such as immune cells or tumor cells. For example,TLR3 activation can trigger various signaling cascades and alter theactivity or expression of numerous regulatory molecules in cells, e.g.,TRIF, MyD88, NFκB, ultimately leading to the secretion of interferon andother cytokines, and associated activities such as NK cell activation,the enhancement of CD8+ T cells, and antigen cross-priming by dendriticcells. Antibodies capable of stimulating TLR3 and triggering an immuneresponse in any of these ways are referred to herein as “activating” or“stimulatory” antibodies. They are useful, e.g., for treating orpreventing a condition caused by a decrease in TLR3-expressing cellactivity or number, or where increased TLR3-expressing cell activity canameliorate, prevent, eliminate, or in any way improve the condition orany symptom thereof. Other antibodies, on the other hand, can inhibitthe activation of TLR3-expressing cells, e.g., they can block thebinding of endogenous ligands such as dsRNA to TLR3, or block theability of TLR3 protein to form homodimers in the presence of dsRNA,thus initiating a signaling cascade. These antibodies are thus referredto as “neutralizing” or “inhibitory” or “blocking” antibodies. Suchantibodies are useful, inter alia, for decreasing TLR3-expressing immunecell activity, e.g., for the treatment or prevention of conditionsinvolving excess TLR3-expressing cell activity or number, or wheredecreased TLR3-expressing cell activity can ameliorate, prevent,eliminate, or in any way improve the condition or any symptom thereof.

In a preferred embodiment, the antibodies are capable of stimulatingTLR3 receptors on tumor cells, e.g., breast cancer cells, melanomacells, hepatoma cells, or cervical cancer cells, causing their death viaapoptosis. As such, the antibodies can be used in the treatment of thecancer, e.g., breast cancer, cervical cancer, hepatoma, or melanoma,e.g., by administering the antibodies systemically or topically to thesite of the cancer. Typically, in such embodiments, the expression ofTLR3 in the cancer cells will be assessed in a prior step according tothe present methods, e.g., using the present antibodies to detect TLR3expression, e.g., in a biopsy or other sample of the cancerous tissue orcells.

Any of a large number of assays, including molecular, cell-based, andanimal-based models, can be used to assess the ability of anti-TLR3antibodies to modulate TLR3-expressing cell activity. For example,cell-based assays can be used in which cells expressing TLR3 are exposedto dsRNA, polyIC, or poly AU, or another ligand (or cells expressing theligand), and the ability of the antibody to disrupt the binding of theligand or the stimulation of the receptor (as determined, e.g., byexamining any of the TLR3 cell activities addressed herein, such asinterferon expression, NFκB activity, NK cell activation, etc.) isassessed.

The activity of TLR3-expressing cells can also be assessed in theabsence of a ligand, by exposing the cells to the antibody itself andassessing its effect on any aspect of the cells' activity or behavior.In such assays, a baseline level of activity (e.g., cytokine production,proliferation, see below) of the TLR3-expressing cells is obtained inthe absence of a ligand, and the ability of the antibody or compound toalter the baseline activity level is detected. In one such embodiment, ahigh-throughput screening approach is used to identify compounds capableof affecting the activation of the receptor.

Any suitable physiological change that reflects TLR3 activity can beused to evaluate test antibodies or antibody derivatives. For example,one can measure a variety of effects, such as changes in gene expression(e.g., NFκB-responding genes), protein secretion (e.g., interferon),cell growth, cell proliferation, pH, intracellular second messengers,e.g., Ca²⁺, IP3, cGMP, or cAMP, or activity such as ability to activateNK cells. In one embodiment, the activity of the receptor is assessed bydetecting production of TLR3-responsive cytokines.

In another embodiment, the effect of the present antibodies onTLR3-expressing cells is assessed in non-human primates in vivo. Forexample, a pharmaceutical composition comprising an anti-TLR3 antibodyof the present invention is administered to a non-human primate that iseither healthy or affected by a TLR3-associated condition, e.g. a viralinfection (in which activation of TLR3 could trigger an immune responseand help fight the infection) or cancer (in which activation of TLR3 ontumor cells could lead to apoptosis of the cells), and the effect of theadministration on, e.g., the number or activity of TLR3-expressing cellsin the primate, or on the progression of the condition is assessed. Anyantibody or antibody derivative or fragment that effects a detectablechange in any of these TLR3-related parameters is a candidate for use inthe herein-described methods.

In any of the herein-described assays, an increase or decrease of 5%,10%, 20%, preferably 30%, 40%, 50%, most preferably 60%, 70%, 80%, 90%,95%, or greater in any detectable measure of TLR3-stimulated activity inthe cells indicates that the test antibody is suitable for use in thepresent methods.

Compositions and Uses in Diagnostics, Prognostics and Therapy

As demonstrated herein, the antibodies of the invention are particularlyeffective at detecting cells which express TLR3 polypeptides (e.g.melanomas, breast cancers, etc.), and without non-specific staining ontissues that do not express TLR3 polypeptides. The antibodies willtherefore have advantages for use in the diagnosis, prognosis and/orprediction of pathologies involving TLR3-expressing cells. For example,cancer in patients can be characterized or assessed using an antibody ofthe invention. This can be useful to determine whether a patient has apathology characterized by cells which express TLR3 polypeptides. Themethod can also be useful to determine whether a patient having suchpathology can be treated with a therapy effective in cells which expressTLR3. For example the method can be used to determine if a patient willrespond to an antigen binding compound that binds TLR3 (e.g. anyantibody) or that modulates (e.g. activates or inhibits) TLR3 or aTLR3-linked signaling pathway.

The antibodies described herein can therefore be used for the detection,preferably in vitro, of the presence of TLR3-expressing cells,optionally of a pathology where TLR3-expressing cells are present (e.g.cancer, infection, inflammatory or autoimmune disorders). Such a methodwill typically involve contacting a biological sample (e.g.paraffin-embedded tissue section) from a patient with an antibodyaccording to the invention and detecting the formation of immunologicalcomplexes resulting from the immunological reaction between the antibodyand the biological sample. The complex can be detected directly bylabelling the antibody according to the invention or indirectly byadding a molecule which reveals the presence of the antibody accordingto the invention (secondary antibody, streptavidin/biotin tag, etc.).For example, labelling can be accomplished by coupling the antibody withradioactive or fluorescent tags. These methods are well known to thoseskilled in the art. Accordingly, the invention also relates to the useof an antibody according to the invention for preparing a diagnosticcomposition that can be used for detecting the presence ofTLR3-expressing cells, optionally for detecting the presence of apathology where TLR3-expressing cells are present, optionally forcharacterizing a cancer or other pathology, in vivo or in vitro.

In some embodiments, the antibodies of the invention will also be usefulfor predicting a cancer prognosis; a cancer prognosis, a prognostic forcancer or cancer progression comprises providing the forecast orprediction of (prognostic for) any one or more of the following:duration of survival of a subject susceptible to or diagnosed with acancer, duration of recurrence-free survival, duration of progressionfree survival of a subject susceptible to or diagnosed with a cancer,response rate to treatment in a subject or group of subjects susceptibleto or diagnosed with a cancer, and/or duration of response, degree ofresponse, or survival following treatment in a subject.

The antibodies of the invention will also be generally useful fordetermining whether a subject is suitable for, or for predicting theresponse of a subject to, treatment with a therapeutic agent directed toa TLR3-expressing cell. Preferably the therapeutic agent is anantigen-binding fragment (e.g. an antibody, an antibody of theinvention) that binds to or modulates (e.g. activates or inhibits) aTLR3 polypeptide.

The antibodies of the invention will also be useful, for example, forassessing the response of a subject having cancer to a treatment with aTLR3 ligand, e.g., a TLR3 agonist such as a dsRNA, polyIC, polyAU, or anactivating anti-TLR3 antibody; such a method will typically involveassessing whether the patient has cancer cells that express a TLR3polypeptide bound by an antibody of the invention, the expression ofTLR3 polypeptide being indicative of a responder subject. A positivedetermination that a patient has cancer cells that express TLR3polypeptides indicates that the patient will be a positive responder totreatment with the TLR3 ligand.

Also encompassed is a diagnostic or prognostic kit, in particular forcancer, comprising an antibody according to the invention. Optionallythe kit comprises an antibody of the invention for use as a diagnosticor prognostic, and a TLR3 ligand (e.g. a TLR3 modulating agent, a TLR3agonist or a TLR3 antagonist), optionally wherein the TLR3 ligand is anantibody or nucleic acid (e.g. dsRNA), for use as a therapeutic. Saidkit can additionally comprise means by which to detect the immunologicalcomplex resulting from the immunological reaction between the biologicalsample and an antibody of the invention, in particular reagents enablingthe detection of said antibody.

In preferred embodiments, the present antibodies are used in diagnostic,or combined diagnostic-therapeutic, methods, involving the detection ofTLR3 in cells present on paraffin-embedded tissue samples. Inparticularly preferred embodiments, the tissue samples comprise tumortissue, e.g., from breast, melanoma, lung, esophagus, stomach, larynx,kidney, or cervix. Typically, in such embodiments, a paraffin-embeddedslide comprising tissue obtained from a biopsy from the patient will beprovided, and the presence of TLR3 in the tissue will be assessed usingthe herein-described antibodies and methods. A detection of TLR3 in thetissue sample indicates that the patient is suitable for treatment witha TLR3 ligand, e.g., a TLR3 agonist such as a dsRNA, polyIC, polyAU, oran activating anti-TLR3 antibody. Preferably the TLR3 ligand is an agentcapable of inducing apoptosis of TLR3-expressing cells (e.g. polyAU,polyIC). In other embodiments, the method may further comprise theadditional step of administering to said patient the TLR3 ligand. Inother embodiments, the method may further comprise the additional stepof administering to said patient, in combination or addition to the TLR3ligand, an appropriate additional therapeutic agent selected from animmunomodulatory agent, a hormonal agent, a chemotherapeutic agent, ananti-angiogenic agent, an apoptotic agent, a second antibody that bindsto and modulates a TLR3 receptor, an anti-infective agent, a targetingagent, an anti-inflammation drug, a steroid, an immune systemsuppressor, an antibiotic, or an adjunct compound.

Certain compositions can also be used to induce the expression of TLR3in cells, including TLR3 agonists (e.g. dsRNA such as polyAU) andIFN-alpha. The present antibodies are also useful in a method ofsensitizing a cell or a patient to a treatment, e.g. with a TLR3agonist, and/or to detect if a patient (or cell) is sensitized totreatment. Such methods can be useful in the treatment of cancers inparticular. Typically, in such embodiments, the method comprisescontacting a biological sample (e.g. a tumor sample) with, oradministering to the patient, an effective amount of a compositioncapable of inducing TLR3 expression in a cell, providing aparaffin-embedded slide comprising tissue obtained from a biopsy fromthe patient, and assessing the presence of TLR3 in the tissue using theherein-described antibodies and methods. A detection of TLR3 in thetissue sample indicates that the patient has been sensitized to atreatment, e.g. with a TLR3 ligand. In other embodiments, the method mayfurther comprise the additional step of administering to said patientthe TLR3 ligand.

The present invention also provides pharmaceutical compositions thatcomprise an antibody, or a fragment and derivative thereof, wherein saidantibody, fragment or derivative specifically binds to TLR3 polypeptideson the surface of cells, and optionally modulates the activity ofTLR3-expressing cells comprising the polypeptides and, consequently, theactivity or behavior of the cells expressing the polypeptides, e.g.,TLR3-expressing tumor or immune cells. In certain embodiments, theantibodies stimulate the TLR3 and thus enhance the activity orproliferation of the cells, or induce their apoptosis. In otherembodiments, the antibodies inhibit the TLR3, e.g., by blocking theinteraction of an antigen or ligand such as dsRNA to the receptor, andthus inhibits the proliferation or activation of the cells. Thecomposition further comprises a pharmaceutically acceptable carrier.Such compositions are also referred to as “antibody compositions” of theinvention. In one embodiment, antibody compositions of this inventioncomprise an antibody disclosed in the antibody embodiments above. Theantibody 40F9 is included within the scope of antibodies that may bepresent in the antibody compositions of this invention.

The invention further provides a method of modulating TLR3-expressingcell activity in a patient in need thereof, comprising the step ofadministering to said patient a composition according to the invention.In one embodiment, the cell activity is enhanced, wherein the patienthas a disease or disorder wherein such enhancement may promote, enhance,and/or induce a therapeutic effect (or promotes, enhances, and/orinduces such an effect in at least a substantial proportion of patientswith the disease or disorder and substantially similar characteristicsas the patient, as may determined by, e.g., clinical trials). Includedin such embodiments are methods in which the antibodies are administeredto induce the apoptosis of TLR3-expressing tumor cells. In anotherembodiment, the TLR3-expressing cell activity is inhibited, wherein thepatient has a disease or disorder wherein such inhibition may promote,enhance, and/or induce a therapeutic effect (or promotes, enhances,and/or induces such an effect in at least a substantial proportion ofpatients with the disease or disorder and substantially similarcharacteristics as the patient—as may determined by, e.g., clinicaltrials).

In other embodiments, the method may comprise the additional step ofadministering to said patient an appropriate additional therapeuticagent selected from an immunomodulatory agent, a hormonal agent, achemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, asecond antibody that binds to and modulates a TLR3 receptor, ananti-infective agent, a targeting agent, an anti-inflammation drug, asteroid, an immune system suppressor, an antibiotic, or an adjunctcompound. Such additional agents can be administered to said patient asa single dosage form together with said antibody, or as a separatedosage form. The dosage of the antibody (or antibody and the dosage ofthe additional therapeutic agent collectively) are sufficient todetectably induce, promote, and/or enhance a therapeutic response in thepatient. Where administered separately, the antibody, fragment, orderivative and the additional therapeutic agent are desirablyadministered under conditions (e.g., with respect to timing, number ofdoses, etc.) that result in a detectable combined therapeutic benefit tothe patient.

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The antibodies of this invention may also be employed in a method ofmodulating, e.g. enhancing or inhibiting, the activity ofTLR3-expressing cells in a patient or a biological sample. This methodcomprises the step of contacting said composition with said patient orbiological sample. Such method will be useful for both diagnostic andtherapeutic purposes.

For use in conjunction with a biological sample, the antibodycomposition can be administered by simply mixing with or applyingdirectly to the sample, depending upon the nature of the sample (fluidor solid). The biological sample may be contacted directly with theantibody in any suitable device (plate, pouch, flask, etc.). For use inconjunction with a patient, the composition must be formulated foradministration to the patient.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

Sterile injectable forms of the compositions of this invention may beaqueous or an oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents that are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The compositions of this invention may be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, aqueous suspensions or solutions. In the case of tablets fororal use, carriers commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude, e.g., lactose. When aqueous suspensions are required for oraluse, the active ingredient is combined with emulsifying and suspendingagents. If desired, certain sweetening, flavoring or coloring agents mayalso be added.

Alternatively, the compositions of this invention may be administered inthe form of suppositories for rectal administration. These can beprepared by mixing the agent with a suitable non-irritating excipientthat is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

The compositions of this invention may also be administered topically,especially when the target of treatment includes areas or organs readilyaccessible by topical application, including diseases of the eye, theskin, or the lower intestinal tract. Suitable topical formulations arereadily prepared for each of these areas or organs. For topicalapplications, the compositions may be formulated in a suitable ointmentcontaining the active component suspended or dissolved in one or morecarriers. Carriers for topical administration of the compounds of thisinvention include, but are not limited to, mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thecompositions can be formulated in a suitable lotion or cream containingthe active components suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers include, but arenot limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol andwater.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Patches may also be used.

The compositions of this invention may also be administered by nasalaerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other conventional solubilizing or dispersingagents.

Several monoclonal antibodies have been shown to be efficient inclinical situations, such as Rituxan Herceptin (Trastuzumab) or Xolair(Omalizumab), and similar administration regimens (i.e., formulationsand/or doses and/or administration protocols) may be used with theantibodies of this invention. Schedules and dosages for administrationof the antibody in the pharmaceutical compositions of the presentinvention can be determined in accordance with known methods for theseproducts, for example, using the manufacturers' instructions. Forexample, an antibody present in a pharmaceutical composition of thisinvention can be supplied at a concentration of 10 mg/mL in either 100mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulatedfor IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodiumcitrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water forInjection. The pH is adjusted to 6.5. An exemplary suitable dosage rangefor an antibody in a pharmaceutical composition of this invention maybetween about 1 mg/mL and 500 mg/mL. However, it will be appreciatedthat these schedules are exemplary and that an optimal schedule andregimen can be adapted taking into account the affinity and tolerabilityof the particular antibody in the pharmaceutical composition that mustbe determined in clinical trials. Quantities and schedule of injectionof an antibody in a pharmaceutical composition of this invention thatsaturate TLR3-expressing cells for 24 hours, 48 hours, 72 hours, or aweek or a month will be determined considering the affinity of theantibody and its pharmacokinetic parameters.

According to another embodiment, the antibody compositions of thisinvention may further comprise another therapeutic agent, includingagents normally utilized for the particular therapeutic purpose forwhich the antibody is being administered. The additional therapeuticagent will normally be present in the composition in amounts typicallyused for that agent in a monotherapy for the particular disease orcondition being treated. Such therapeutic agents include, but are notlimited to, therapeutic agents used in the treatment of cancers,therapeutic agents used to treat infectious disease, therapeutic agentsused in other immunotherapies, cytokines (such as IL-2 or IL-15),anti-inflammation agents, steroids, immune system suppressors,antibiotics, and other antibodies and fragments thereof.

In another embodiment, two or more antibodies of this invention havingdifferent cross-reactivities, e.g. antibodies that specifically bind todistinct epitopes within the TLR3 polypeptide on paraffin-embeddedtissue samples, are combined in a single composition so as to target asmany distinct TLR3 gene products as possible, e.g. to account fordiversity in the polypeptides within an individual or in differentpatients, and to do so as efficaciously as possible. In addition, anantibody composition of this invention may comprise multiple antibodiesthat recognize a single TLR3 epitope. Such combinations would againprovide wider utility in a therapeutic setting or ensure optimal TLR3staining in paraffin embedded tissue sections.

The invention also provides a method of modulating TLR3-expressing cellactivity in a patient in need thereof, comprising the step ofadministering a composition according to this invention to said patient.The method is more specifically directed at increasing TLR3 cellactivity in patients having a disease in which increased TLR3 cellactivity is beneficial (e.g., infection such as a viral infection, orcancer in which the cancer cells express TLR3), or which is caused orcharacterized by insufficient TLR3 cell activity, or, contrarily, atdecreasing TLR3 cell activity in patients having a disease in whichdecreased TLR3 cell activity is beneficial, or which is caused orcharacterized by excessive TLR3 cell activity.

Diseases and conditions in which the present methods can be used includecancer, other proliferative disorders, infectious disease, or immunedisorders such as inflammatory diseases and autoimmune diseases. Morespecifically, the methods of the present invention are utilized for thetreatment of a variety of cancers and other proliferative diseasesincluding, but not limited to, carcinoma, including that of the bladder,breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach,cervix, thyroid and skin, including squamous cell carcinoma;hematopoietic tumors of lymphoid lineage, including leukemia, acutelymphocytic leukemia, acute leukemia, B-cell lymphoma, T-cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma andBurkett's lymphoma; hematopoietic tumors of myeloid lineage, includingacute and chronic myelogenous leukemias and promyelocytic leukemia;tumors origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma andglioma; tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors origin,including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and othertumors, including melanoma, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma. In preferred embodiments, themethods are used to diagnose or treat tumors selected from the groupconsisting of breast, melanoma, lung, esophagus, stomach, larynx,kidney, and cervix. In preferred embodiments, the cancer comprisesTLR3-expressing cells, and the present antibodies and methods are usedto detect TLR3 expression in the cells.

Other proliferative disorders in which the present methods can be usedinclude for example hyperplasias, fibrosis (especially pulmonary, butalso other types such as renal fibrosis), angiogenesis, psoriasis,atherosclerosis and smooth muscle proliferation in the blood vessels,such as stenosis or restenosis following angioplasty.

The antibodies of this invention can be used to treat or preventinfectious diseases, e.g., by detecting TLR3 levels in immune cells invitro or ex vivo, and subsequently inducing TLR3 activity (in the caseof a detection of TLR3) to activate the immune system. Targetedinfections are preferably any infections caused by viruses, bacteria,protozoa, molds or fungi. Such viral infectious organisms include, butare not limited to, hepatitis type A, hepatitis type B, hepatitis typeC, influenza, varicella, herpes simplex type I (HSV-1), herpes simplextype 2 (HSV-2), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papilloma virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, and humanimmunodeficiency virus type I HIV-2).

Bacterial infections that can be treated according to this inventioninclude, but are not limited to, infections caused by the following:Staphylococcus; Streptococcus, including S. pyogenes; Enterococci;Bacillus, including Bacillus anthracis; Lactobacillus; Listeria;Corynebacterium diphtheriae; Gardnerella, including G. vaginalis;Nocardia; Streptomyces; vulgaris; Pseudomonas, including aeruginosa;Legionella; Neisseria, including N. meningitides; Flavobacterium,including F. meningosepticum; Brucella; Bordetella, including B.pertussis and B. bronchiseptica; Escherichia, including E. coli;Enterobacter; Serratia, including S. marcescens and S. liquefaciens;Edwardsiella; Proteus, including P. mirabilis and P. vulgaris;Streptobacillus; Rickettsiaceae, including R. rickettsii; Chlamydia,including C. psittaci; Mycobacterium, including M. tuberculosis, M.intracellulare, M. folluiturn, M. laprae, M. avium, M. bovis, and M.kansasii; and Nocardia.

Protozoa infections that may be treated according to this inventioninclude, but are not limited to, infections caused by leishmania,kokzidioa, and trypanosoma. A complete list of infectious diseases canbe found on the website of the National Center for Infectious Disease(NCID) at the Center for Disease Control (CDC), which list isincorporated herein by reference.

Immune disorders treatable using the present methods include, interalia, autoimmune disorders and inflammatory disorders, including, butnot limited to, Crohn's disease, Celiac disease, ulcerative colitis,irritable bowel syndrome, acute disseminated encephalomyelitis (ADEM),Addison's disease, antiphospholipid antibody syndrome (APS), aplasticanemia, autoimmune hepatitis, Diabetes mellitus, Goodpasture's syndrome,Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease,lupus erythematosus, Multiple sclerosis, Myasthenia gravis, opsoclonusmyoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus,primary biliary cirrhosis, psoriasis, rheumatoid arthritis, Reiter'ssyndrome, Takayasu's arteritis, temporal arteritis, warm autoimmunehemolytic anemia, Wegener's granulomatosis, appendicitis, arteritis,arthritis, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis,cholangitis, cholecystitis, chorioamnionitis, colitis, conjunctivitis,cystitis, dacryoadenitis, dermatitis, dermatomyositis, encephalitis,endocarditis, endometritis, enteritis, enterocolitis, epicondylitis,epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis,gingivitis, hepatitis, hidradenitis suppurativa, ileitis, iritis,laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis,nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis,pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis,pleuritis, phlebitis, pneumonitis, proctitis, prostatitis,pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis,tendonitis, tonsillitis, uveitis, vaginitis, vasculitis, and vulvitis.

The present antibodies can be included in kits, which may contain anynumber of antibodies and/or other compounds, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or any other number of therapeutic antibodies and/orcompounds, as well as, in certain embodiments, antibodies or otherdiagnostic reagents for detecting the presence of TLR3 inparaffin-embedded tissue samples. Such diagnostic antibodies will oftenbe labeled, either directly or indirectly (e.g., using secondaryantibodies, which are typically included in the kit, together withreagents, e.g., buffers, substrates, necessary for their detection).Therapeutic antibodies can be either modified, e.g. by the addition of acytotoxic agent, or unmodified, working, e.g., by modulating TLR3activation, or by simply binding to target cells and thereby stimulatingor inhibiting them, triggering cell death, or marking them fordestruction by the immune system. It will be appreciated that thisdescription of the contents of the kits is not limiting in any way. Forexample, the kit may contain other types of therapeutic compounds aswell, such as other anti-tumor agents. Preferably, the kits also includeinstructions for using the antibodies, e.g., detailing theherein-described methods.

Further aspects and advantages of this invention will be disclosed inthe following experimental section, which should be regarded asillustrative and not limiting the scope of this application.

EXAMPLES Materials and Methods

Interferon-alpha (IntronA™) was purchased from Schering Plough Corp.Tumor Cell lines: A375 malignant melanoma tumor cell lines (CRL-1619)are purchased from ATCC. Antibodies (antigen, supplier, reference):Anti-TLR3 antibody pAb, R&D Systems, ref. AF1487. Instrumentation:FACSCalibur™ flow cytometer (BD Biosciences).

Inhibition with lentivirus shRNAA lentivirus construction was made andproduced by Vectalys (Toulouse, France), encoding short hairpin RNA(shRNA) targeting control human TLR3. Tumor cells were infected withlentivirus preparation and further selected with puromycin to get stableshTLR3A375 tumor cells.

Surface Plasmon resonance (SPR)(a) Biacore T100 general procedure. SPRmeasurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments HBS-EP+ buffer (BiacoreGE Healthcare) served as running buffer and sensorgrams were analyzedwith Biaevaluation 4.1 and Biacore T100 Evaluation software. Recombinanthuman TLR3 and TLR4 were purchased from R&D Systems.

(b) Protein immobilization. Recombinant TLR3 and TLR4 proteins wereimmobilized covalently to carboxyl groups in the dextran layer of aBiacore Sensor Chip CM5 (chip). The chip surface was activated withEDC/NHS (0.2M N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride, 0.05M N-hydroxysuccinimide (Biacore GEHealthcare)). Proteins were diluted to 10 μg/ml in coupling buffer (10mM sodium acetate, pH 5.6) and injected until the appropriateimmobilization level was reached (i.e. approximately 2000 RU).Deactivation of the remaining activated groups was performed using 100mM ethanolamine pH 8 (Biacore GE Healthcare).(c) Antibody binding analysis. Antibodies at a constant concentration of10 μg/ml were injected for 2 min at a constant flow rate of 10 μl/minover the immobilized proteins and allowed to dissociate for 3 min beforeregeneration by a ten second injection of 10 mM NaOH, 500 mM NaClregeneration buffer. Blank correction is performed on line byco-injecting the soluble antibodies onto the reference dextran flowcell.

Example 1 Comparison with Commercially Available Antibodies

Commercially available anti-TLR3 antibodies were tested in animmunocytochemistry (ICC) assay on 293T and 293-TLR3 cells. Briefly,cells were coated on Marienfeld adhesion slides, (#80107926, VWR) andfixed in cold acetone for 20 nm at 4° C. Cells were permeabilized withpermwash (#554723, from BD biosciences) and anti-TLR3 antibodies,labeled with biotin, followed by streptavidine coupled to peroxidasefrom SIGMA. Staining was revealed with AEC. TLR3.7 antibody yieldedfaint staining on 293T-TLR3 cells overexpressing TLR3, but no stainingon other TLR3 expressing tumor cells. The goat pAb anti-TLR3Ab from R&Dsystems yielded strong staining on 293T-TLR3, specific since no stainingcould be obtained on control 293T cells.

The two antibodies were tested in Biacore for binding to immobilizedhuman TLR3 and TLR4 protein. TLR3.7 antibody showed low binding to TLR3but not TLR4. Likewise, the goat pAb anti TLR3 from R&D systems whichshowed staining in paraffin-embedded sample, albeit to TLR4 positivesamples in addition to TLR3, showed a high response for binding to TLR3but not TLR4 (see FIG. 1).

Finally several commercially available anti-TLR3 antibodies (reported intheir technical datasheet to work mostly in WB assay, but not in FACS orIHC) were tested in a series of assays. The antibodies were tested in anIHC assays for binding to 293T cells, 293T-TLR3 cells and 293T-TLR4cells, for cells in either frozen cell pellets or in paraffin-embeddedcell pellets. Results are shown in Table 1.

TABLE 1 Frozen cell pellet Paraffin-embedded cell pellet α h-TLR3Supplier 293T 293T.T4 293T.T3 293T 293T.T4 293T.T3 Goat pAb R&D systems− − + + + + 40C1285.6 Imgenex + + + + + + TLR3.7 eBiosciences − − − − −− MCA2267 Abd Serotec − − − − + + A01 Abnova − − − − − − Q-18 Santa Cruz− − − − − − N-14 Santa Cruz − − + − − − L-13 Santa Cruz − − − − − − C-20Santa Cruz − − + − − − H-125 Santa Cruz − − − − − −

In conclusion, although some mAb or pAb are able to detect human TLR3 inICC, FACS or frozen cell pellet. Among them, only one goat pAb from R&Dwas able to induce a signal strong enough to stain normal human tissuesection with endogenous expression of TLR3 (as opposed to TLR3overexpressing 293T cells transfected with TLR3). In addition, none ofthe commercially available antibodies were able to detect human TLR3 inparaffin-embedded cell pellets or tissue section, with specificity forTLR3 over TLR4.

Example 2 Obtaining of TLR3—Specific Monoclonal Antibodies

Primary Screen.

To obtain anti-TLR3 antibodies, Balb/c mice were immunized with arecombinant human His-tagged TLR3 extracellular domain recombinantprotein (R&D systems, #1487-TR-050). Mice received oneprimo-immunisation with an emulsion of 50 μg TLR3 protein and CompleteFreund Adjuvant, intraperitoneally, a 2nd immunization with an emulsionof 50 μg TLR3 protein and Incomplete Freund Adjuvant, intraperitoneally,and three boosts with 10 μg TLR3 protein, intravenously. Immune spleencells were fused with X63.Ag8.653 immortalized B cells, and cultured inthe presence of irradiated spleen cells. 40 culture plates were obtainedand evaluated in a first screen for TLR3 binding using an ELISAdeveloped for detection of binding to TLR3. Briefly, His-taggedrecombinant TLR3 protein (R&D systems, #1487-TR-050) was coated onNi-NTA 96-wells plates (Qiagen). Supernatant (SN) from hybridoma cultureplates and incubated in TLR3-plates, and the presence of TLR3 binding Igwas revealed with goat anti-mouse F(ab) IgG-HRP. Positive supernatantswere selected and tested for lack of binding to TLR4. Briefly,His-tagged rec TLR4 protein (R&D systems, #3146-TR-050) were coated onNi-NTA 96-wells plates (Qiagen). SN from hybridoma culture plates wereincubated in TLR4-plates, and the presence of TLR4 binding Ig wasrevealed with goat anti-mouse F(ab) IgG-HRP. TLR4 was chosen as a 2^(nd)screen in order to discriminate among wells selected in the 1^(st)screen, where anti-His specific antibody from TLR3 specific antibodywere used. Secondly, given the homology between TLR3 and other membersof TLR family, the initial assessment in Example 1 demonstrated that atleast one commercially available monoclonal antibody (mAb) indicated onits packaging as specific for TLR3 protein nevertheless recognizedparaffin-embedded 293T cells stably transfected with TLR4 (Results foravailable commercial antibodies are shown in Example 1).

Secondary Screen; Selection of Hybridomas of Interest.

168 supernatants were retained and tested in a further screen in aBiacore assay using rec TLR3 chips, followed by various assays formatsbased on binding to human TLR3-expressing 293T cells. A 293T cell line(ATCC, #CRL-1573), stably transfected with pISRE-luc plasmid(#219089—Stratagene), was further selected as inducing optimal responseto IFN-alpha stimulation and referred to as control 293T cells. Thiscell line was further stably transfected with pUNO-hTLR3 plasmid(#puno-htlr3—InVivogen), or pUNO-hTLR4 plasmid (#puno-tlr4—InVivogen)and referred to respectively as 293T-TLR3 and 293T-TLR4. Supernatantswere screened in a FACS based screen for binding to 293T-TLR3 cells withno binding to 293T control cells, and in parallel in an IHC screen forbinding to 293T-TLR3 cells as a frozen cell pellet, with no binding to293T-TLR4 cells. Wells selected in the IHC screen for binding to293T-TLR3 cells as a frozen cell pellet were also further tested in anIHC screen for binding to 293T-TLR3 cells as a paraffin embedded cellpellet. Briefly, for FACS screening, the presence of reacting antibodiesin supernatants were revealed by Goat anti-mouse polycolonal antibody(pAb) labeled with biotin, streptavidin labeled with PE. For IHCscreening, presence of reacting antibodies (Abs) in supernatants wererevealed by donkey anti-mouse pAb labeled with biotin (#715-065-150,Jackson Immunoresearch Laboratories), streptavidin labeled withperoxydase (#E2886, SIGMA) and revealed with DAB (#SK-4105, VectorLaboratories). Seven supernatants tested positive in this screen forbinding to 293T-TLR3 cells in a paraffin embedded cell pellet.

Cloning of Hybridomas of Potential Interest.

42 potentially interesting hybridomas selected from the initialscreening were cloned by limiting dilution techniques in 96-wellsplates, and 304 subclones were tested in a series of screens as follows.The 304 subclones were first evaluated in a screen for TLR3 bindingusing the same ELISA developed for detection of binding to TLR3, andpositive supernatants were selected and tested for lack of binding toTLR4 in ELISA assay, yielding 228 clones which were selective for TLR3.All supernatants yielding a ratio above 10 for DO obtained in TLR3ELISAto DO obtained in TLR4ELISA were selected as specific for TLR3. Amongthem was supernatant from well F9 of plate 40 (40F9).

Among the 304 clones, 63 clones, selected as issuing from preclonestested positive in frozen IHC, were also tested in a frozen IHC screenfor binding to 293T-TLR3 cells as a frozen cell pellet, with no bindingto 293T-TLR4 cells, yielding 31 positive clones in frozen IHC.

Among 71 clones positive in FACS staining and the 31 clones positives infrozen IHC, 60 clones were selected for cryopreservation from the 304initial clones. These 60 selected clones were also tested in an IHCscreen for binding to 293T-TLR3 cells as a paraffin embedded cell pelletand 6 clones were positive for TLR3 binding. Those clones were furthertested for binding to human healthy colon tissue embedded in paraffin(#T8235722-5, Biochain), previously reported to be positive for TLR3expression. Three of them stained positively human colon tissue, all ofthem issuing for the same pre-clone, 40F9. The clone 40F9.6 yielded thestrongest signal and was selected for further TLR3 expression study.

Example 3 Development of an Amplification System and Staining Protocolfor 40F9

40F9.6 was tested on tonsil tissue with different detection systems, inorder to identify the detection system yielding the strongest signal,with the lowest noise:signal ratio. The ENVISION System commercializedby DAKO A/S (Glostrup, Denmark) was selected. ENVISION is anamplification system based on an HRP-labeled polymer conjugated tosecondary antibodies. Tissue sections were dewaxed and rehydrated beforethe antigen unmasking procedure in boiled citrate buffer (pH6).Endogenous peroxydases were blocked by a 3% solution of H₂O₂. 40F9.6, at10 μg/ml, was applied on tissue sections, incubated for 1 hour at roomtemperature, and was revealed by using the ENVISION System andDAB-chromogen (Dako, Glostrup, Denmark).

Example 4 Testing Antibodies in Paraffin-Embedded Tissue Sections fromCancer Patients

40F9.6 was tested in a series of paraffin-embedded samples. In a firstset of experiments, purified 40F9.6 was tested in an IHC screen forbinding to 293T-TLR3 and 293T-TLR4 cells as a paraffin embedded cellpellet from 293T-TLR3 transfectants tissue, at 10 μg/ml, with theamplification protocol described in Example 3. 40F9.6 showed staining on293T-TLR3 cells but no staining on 293T-TLR4 cells.

In a second set of experiments, purified 40F9.6 was tested in an IHCscreen for binding to human A375 melanoma cells treated withinterferon-α, in paraffin embedded cell pellet, again at 10 μg/ml, withthe amplification protocol described in Example 3. A375 cells werepreviously identified as TLR3 positive, and interferon-α is reported toadditionally enhance TLR3 expression. A control IgG1 antibodies was usedfor comparison. 40F9 showed staining on A375 cells while control IgG1showed no staining The same experiment was also carried out ininterferon-α treated A375 cells which had been stably infected with alentiviral construction coding for shRNA targeting TLR3. Staining by40F9.6 but not IgG1 decreased substantially in the A375 cells treatedwith shRNA.

In a further set of experiments, purified 40F9.6 was tested in an IHCscreen for binding to various healthy human tissue in paraffin embeddedtissue sections, again at 10 μg/ml, with the amplification protocoldescribed in Example 3, using control IgG1 antibodies was used forcomparison. 40F9.6 but not IgG1 showed staining in breast, tonsil,esophagus, skin, cerebellum and pancreas tissue. The same experiment wasrepeated in a series of healthy human tissues using antibody PolyR&D(R&D Systems), which has been reported to bind human TLR3. Both 40F9.6and PolyR&D antibodies stained skin, cerebellum, breast, lung,esophagus, stomach, ileum, jejunum, duodenum, colon, liver, pancreas,testis, spleen, thymus thyroid and kidney.

In another set of experiments, 40F9.6 was tested in an IHC screen forbinding to various human tumor tissues in paraffin embedded tissuesections, a standard donkey anti-mouse biotin amplification, instead ofthe Envison™ system. 10 tumor samples were used for each tumorindication. 40F9.6 showed strong staining in several tumor types,including lung (6 of 10 samples), esophagus (8 of 10 samples), stomach(4 of 10 samples), larynx (4 of 10 samples) and cervix (7 of 10samples).

In another set of experiments, purified 40F9.6 was tested in an IHCscreen for binding to melanoma tissue samples in paraffin embeddedtissue sections from 9 human patients. Melanomas were generally frompatients who either did or did not receive interferon-α treatment priorto biopsy. 40F9.6 was used at 10 μg/ml, using both the standard biotineamplification and Envision™ amplification systems. 40F9.6 showedstaining in 8 or 9 patients, ranging in degree of staining, and stainingin patients both who received or did not undergo interferon-α treatment.The patients not treated with interferon-α represented three of theeight patients; one of these non-treated patients showed strong stainingwith 40F9.6, one showed no staining and one showed a low level ofstaining.

In these experiments, 40F9.6 clone appears to stain specifically TLR3 ina great variety of paraffin embedded tissues, with a selectivity higherthan any of the currently available TLR3 antibodies.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way. Anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents, Thedescription herein of any aspect or embodiment of the invention usingterms such as reference to an element or elements is intended to providesupport for a similar aspect or embodiment of the invention that“consists of” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e.g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law. All publications and patentapplications cited in this specification are herein incorporated byreference in their entireties as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

We claim:
 1. A method of treating a cancer patient, said methodcomprising: a) providing a paraffin-embedded tissue section of canceroustissue from a patient having melanoma breast cancer, lung cancer,esophageal cancer, stomach cancer, cancer of the larynx, kidney canceror cervical cancer and detecting TLR3 in said paraffin-embeddedcancerous tissue section with a monoclonal antibody that specificallybinds to TLR3 in paraffin-embedded cancerous tissue sections, saidmonoclonal antibody comprising: i) variable light chain region CDRs:KSSQSLLDSDGKTYLN (SEQ ID NO:5; CDR1); LVSKLDS (SEQ ID NO:6; CDR2); andWQGIHLPYT (SEQ ID NO:7; CDR3) and variable heavy chain region CDRs:YTFTNYGMN (SEQ ID NO:8; CDR1); NANTGEPTYAEEFKG (SEQ ID NO:9; CDR2); andDYDY (SEQ ID NO:10; CDR3); or ii) variable light chain region CDRs:KSSQSLLDSDGKTYLN (SEQ ID NO:5; CDR1); LVSKLDS (SEQ ID NO:6; CDR2); andWQGIHLPYT (SEQ ID NO:7; CDR3) and variable heavy chain region CDRs:KASGYTFTNYGMN (SEQ ID NO:11; CDR1); NANTGEPTYAEEFKG (SEQ ID NO:9; CDR2);and DYDY (SEQ ID NO:10; CDR3); and b) treating a cancer patient positivefor the presence of TLR3 in said paraffin-embedded cancerous tissuesection by administering a TLR3 ligand to said cancer patient.
 2. Themethod of claim 1, wherein the TLR3 ligand is selected from the groupconsisting of a dsRNA, polyIC, polyAU, an activating anti-TLR3 antibodyand an agent capable of inducing apoptosis of TLR3-expressing cells. 3.The method of claim 1, wherein said paraffin-embedded cancerous tissuesection contains breast cancer tissue.
 4. The method of claim 1, whereinsaid paraffin-embedded cancerous tissue section contains lung cancertissue.
 5. The method of claim 1, wherein said paraffin-embeddedcancerous tissue section contains esophageal cancer tissue.
 6. Themethod of claim 1, wherein said paraffin-embedded cancerous tissuesection contains stomach cancer tissue.
 7. The method of claim 1,wherein said paraffin-embedded cancerous tissue section contains larynxcancer tissue.
 8. The method of claim 1, wherein said paraffin-embeddedcancerous tissue section contains kidney cancer tissue.
 9. The method ofclaim 1, wherein said paraffin-embedded cancerous tissue sectioncontains cervical cancer tissue.
 10. The method of claim 1, wherein saidparaffin-embedded cancerous tissue section contains melanoma tissue.